Modulation of CD40 expression

ABSTRACT

Disclosed herein are antisense compounds and methods for decreasing CD40. Examples of disease conditions that can be ameliorated with the administration of antisense compounds targeted to CD40 include hyperproliferative disorders, graft versus host disease (GVHD), graft rejection, asthma, airway hyperresponsiveness, chronic obstructive pulmonary disease (COPD), multiple sclerosis (MS), systemic lupus erythematosus (SLE), and certain forms of arthritis.

RELATED APPLICATIONS

This application is a 35 U.S.C §371 national phase application ofinternational application serial no. PCT/US2008/012998, filed on Nov.20, 2008, which is a non-provisional of and claims priority to U.S.patent application Ser. No. 60/989421, filed on Nov. 20, 2007, thedisclosure of each of which is incorporated herein by reference in itsentirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled33841-513SEQLIST.txt, created Nov. 19, 2008, which is 67 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety. This sequence listingis identical to the sequence listing filed on Nov. 20, 2007, with theexception of the addition of SEQ ID NO: 237.

FIELD OF THE INVENTION

The present invention provides methods and compositions for lowinglevels of CD40 in an animal. Such methods and compositions are useful asanti-inflammatory compounds and anti-tumor compounds.

BACKGROUND OF THE INVENTION

The immune system serves a vital role in protecting the body againstinfectious agents. It is well established, however, that a number ofdisease states and/or disorders are a result of either abnormal orundesirable activation of immune responses. Common examples includegraft versus host disease (GVHD) and graft rejection, and autoimmunelinked diseases such as multiple sclerosis (MS), systemic lupuserythematosus (SLE), and certain forms of arthritis.

In general, an immune response is activated as a result of either tissueinjury or infection. Both cases involve the recruitment and activationof a number of immune system effector cells (e.g., B- and T-lymphocytes,macrophages, eosinophils, neutrophils) in a process coordinated througha series of complex cell-cell interactions. A typical scenario by whichan immune response is mounted against a foreign protein is as follows:foreign proteins captured by antigen presenting cells (APC's) such asmacrophages or dendritic cells are processed and displayed on the cellsurface of the APC. Circulating T-helper cells which express animmunoglobulin that recognizes (i.e. binds) the displayed antigenundergo activation by the APC. These activated T-helpers in turnactivate appropriate B-cell clones to proliferate and differentiate intoplasma cells that produce and secrete humoral antibodies targetedagainst the foreign antigen. The secreted humoral antibodies are free tocirculate and bind to any cells expressing the foreign protein on theircell surface, in effect marking the cell for destruction by other immuneeffector cells. In each of the stages described above, direct cell-cellcontact between the involved cell types is required in order foractivation to occur. (Gruss et al., Leuk. Lymphoma 1989, 24:393). Inrecent years, a number of cell surface receptors that mediate thesecell-cell contact dependent activation events have been identified.Among these cell surface receptors is CD40 and its physiological ligand,CD40 Ligand (CD40L) which is also known as CD154.

CD40 was first characterized as a receptor expressed on B-lymphocytes.It was later found that engagement of B-cell CD40 with CD40L expressedon activated T-cells is essential for T-cell dependent B-cell activation(i.e. proliferation, immunoglobulin secretion, and class switching). Itwas subsequently revealed that functional CD40 is expressed on a varietyof cell types other than B-cells, including macrophages, dendriticcells, thymic epithelial cells, Langerhans cells, and endothelial cells.These studies have led to the current belief that CD40 plays a broadrole in immune regulation by mediating interactions of T-cells withB-cells as well as other cell types. In support of this notion, it hasbeen shown that stimulation of CD40 in macrophages and dendritic resultsis required for T-cell activation during antigen presentation. (Gruss etal., Leuk. Lymphoma, 1997, 24:393). Recent evidence points to a role forCD40 in tissue inflammation as well. Production of the inflammatorymediators IL-12 and nitric oxide by macrophages have been shown to beCD40 dependent. (Buhlmann and Noelle, J. Clin. Immunol., 1996, 16:83).In endothelial cells, stimulation of CD40 by CD40L has been found toinduce surface expression of E-selectin, ICAM-1, and VCAM-1, promotingadhesion of leukocytes to sites of inflammation (Buhlmann and Noelle, J.Clin. Immunol., 1996, 16:83); Gruss et al., Leuk. Lymphoma, 1997,24:393). Finally, a number of reports have documented overexpression ofCD40 in epithelial and hematopoietic tumors as well as tumorinfiltrating endothelial cells, indicating that CD40 may play a role intumor growth and/or angiogenesis as well (Gruss et al., Leuk. Lymphoma,1997, 24:393; Kluth et al., Cancer Res., 1997, 57:891).

Due to the pivotal role that CD40 plays in humoral immunity, thepotential exists that therapeutic strategies aimed at downregulatingCD40 or interfering with CD40 signaling may provide a novel class ofagents useful in treating a number of immune associated disorders,including but not limited to graft-versus-host disease (GVHD), graftrejection, and autoimmune diseases such as multiple sclerosis (MS),systemic lupus erythematosus (SLE), and certain forms of arthritis.Inhibitors of CD40 may also prove useful as anti-inflammatory compounds,and could therefore be useful as treatment for a variety of inflammatoryand allergic conditions such as asthma, rheumatoid arthritis, allograftrejections, inflammatory bowel disease, autoimmune encephalomyelitis,thyroiditis, various dermatological conditions, and psoriasis. Recently,both CD40 and CD154 have been shown to be expressed on vascularendothelial cells, vascular smooth muscle cells and macrophages presentin atherosclerotic plaques, suggesting that inflammation and immunitycontribute to the atherogenic process. That this process involves CD40signaling is suggested by several studies in mouse models in whichdisruption of CD154 (by knockout or by monoclonal antibody) reduced theprogression or size of atherosclerotic lesions. (Mach et al., Nature,1998, 394:200-3; Lutgens et al., 1999, Nat. Med. 5:1313-6).

Finally, as more is learned of the association between CD40overexpression and tumor growth, inhibitors of CD40 may prove useful asanti-tumor agents and inhibitors of other hyperproliferative conditionsas well.

Currently, there are no known therapeutic agents which effectivelyinhibit the synthesis of CD40. To date, strategies aimed at inhibitingCD40 function have involved the use of a variety of agents that disruptCD40/CD40L binding. These include monoclonal antibodies directed againsteither CD40 or CD40L, soluble forms of CD40, and synthetic peptidesderived from a second CD40 binding protein, A20. The use of neutralizingantibodies against CD40 and/or CD40L in animal models has providedevidence that inhibition of CD40 signaling would have therapeuticbenefit for GVHD, allograft rejection, rheumatoid arthritis, SLE, MS,and B-cell lymphoma. (Buhlmann and Noelle, J. Clin. Immunol, 1996,16:83). Clinical investigations were initiated using anti-CD154monoclonal antibody in patients with lupus nephritis. However, studieswere terminated due to the development of thrombotic events. (Boumpas etal., 2003, Arthritis Rheum. 2003, 48:719-27).

Due to the problems associated with the use of large proteins astherapeutic agents, there is a long-felt need for additional agentscapable of effectively inhibiting CD40 function. Antisenseoligonucleotides avoid many of the pitfalls of current agents used toblock CD40/CD40L interactions and may therefore prove to be uniquelyuseful in a number of therapeutic, diagnostic and research applications.U.S. Pat. No. 6,197,584 (Bennett and Cowsert) discloses antisensecompounds targeted to CD40.

SUMMARY OF THE INVENTION

Provided herein are antisense compounds, compositions, and methods forthe treatment and prevention of inflammatory conditions and cancer.

Antisense compounds described herein may be 12 to 30 nucleobases inlength targeted to a CD40 nucleic acid. In certain embodiments, the CD40nucleic acid may be any of the sequences as set forth in GENBANK®Accession No. X60592.1, incorporated herein as SEQ ID NO: 1; GENBANK®Accession No. H50598.1, incorporated herein as SEQ ID NO: 2; GENBANK®Accession No. AA203290.1, incorporated herein as SEQ ID NO: 3; andnucleotides 9797000 to nucleotide 9813000 of GENBANK Accession No.NT_011362.9, incorporated herein as SEQ ID NO: 4, or GENBANK® AccessionNo. BC064518.1, incorporated herein as SEQ ID NO: 237.

The antisense compound may be 12 to 30 nucleobases in length and mayhave a nucleobase sequence comprising at least 8 contiguous nucleobasescomplementary to an equal length portion of an intron region of the CD40gene, selected from the following regions of SEQ ID NO: 4:

(a) positions 11250-12685, corresponding to intron 6;

(b) positions 2943-6367, corresponding to intron 1,

(c) positions 6447-6780, corresponding to intron 2,

(d) positions 6907-7157, corresponding to intron 3,

(e) positions 7305-7673, corresponding to intron 4,

(f) positions 7768-11187, corresponding to intron 5,

(g) positions 12773-12877, corresponding to intron 7, or

(h) positions 12907-13429, corresponding to intron 8,

wherein the remaining part or parts of the antisense compound are atleast 70% complementary to the sequence shown in SEQ ID NO: 4.Preferably, the remaining parts of the antisense compound are at least75%, 80%, 85%, 90%, 95%, 98%, 99%, or, most preferably, 100%complementary to the sequence shown in SEQ ID NO: 4.

Preferably, the antisense compound may comprise at least 8 contiguousnucleobases complementary to an equal length portion of positions 12527to 12685 of SEQ ID NO: 4, which is a region that can be either part ofintron 6, or can be part of an alternative version of exon 7 when adifferent splice acceptor site is selected. Preferably, the antisensecompound has a nucleobase sequence comprising at least 8 contiguousnucleobases of the nucleobase sequence of SEQ ID NO: 208, wherein thenucleobase sequence of the compound is at least 70% complementary to thesequence shown in SEQ ID NO: 4. Preferably, the antisense compound is atleast 75%, 80%, 85%, 90%, 95%, 98%, 99%, or, most preferably, 100%complementary to the sequence shown in SEQ ID NO: 4. More preferably,the antisense compound has the sequence of SEQ ID NO: 208. Even morepreferably, the antisense compound is 20 nucleobases in length andconsists of the nucleobase sequence of SEQ ID NO: 208. Most preferably,the antisense compound is an antisense oligonucleotide 20 nucleotides inlength having the sequence of nucleotides as set forth in SEQ ID NO:208,wherein each cytosine is a 5-methylcytosine, each internucleosidelinkage is a phosphorothioate linkage, nucleotides 1-5 and 16-20 are2′-O-methoxyethyl nucleotides, and nucleotides 6-15 are2′-deoxynucleotides; most preferably the antisense compound is ISIS396236.

In an alternative embodiment, the antisense compound may be 12 to 30nucleobases in length and have a nucleobase sequence comprising at least8 contiguous nucleobases complementary to an equal length portion of aregion of the CD40 gene, corresponding to positions 13662-16001 of SEQID NO: 4, which forms part of exon 9 or a region 3′ to exon 9, whereinthe remaining parts of the antisense compound are at least 70%complementary to the sequence shown in SEQ ID NO: 4. Preferably, thetarget region of the CD40 gene corresponds to positions 13877-14084,even more preferably to positions 13937-13996, of SEQ ID NO: 4.Preferably, the remaining parts of the antisense compound are at least75%, 80%, 85%, 90%, 95%, 98%, 99%, or, most preferably, 100%complementary to the sequence shown in SEQ ID NO: 4.

In yet another alternative embodiment, the antisense compound is 12 to30 nucleobases in length and has a nucleobase sequence complementary tothe sequence shown in SEQ ID NO: 1, starting at position 69 or 70 of SEQID NO: 1, wherein the nucleobase sequence is at least 95% complementaryto the sequence shown in SEQ ID NO: 1. Preferably, the nucleobasesequence is essentially complementary to the sequence shown in SEQ IDNO: 1. More preferably, the nucleobase sequence is selected from thesequences of SEQ ID Nos: 90 and 163. Even more preferably, the antisensecompound has a nucleobase sequence of SEQ ID NO: 90. Even morepreferably, the antisense compound is 18 or 20 nucleobases in length andconsists of the nucleobase sequence of SEQ ID NO: 90 or SEQ ID NO: 163.The antisense compound may be ISIS26163, ISIS396201 or ISIS396278.Preferably, the antisense compound is an antisense oligonucleotide 18nucleotides in length having the sequence of nucleotides as set forth inSEQ ID NO: 90, wherein each cytosine is a 5-methylcytosine, eachinternucleoside linkage is a phosphorothioate linkage, nucleotides 1-4and 15-18 are 2′-O-methoxyethyl nucleotides, and nucleotides 5 to 14 are2′-deoxynucleotides. Most preferably, the antisense compound isISIS26163.

An antisense compound according to the invention may comprise a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 12 contiguous nucleobases of anucleobase sequence selected from among the nucleobase sequences recitedin SEQ ID NOs: 5 to 236. Preferably, the compound consists of asingle-stranded modified oligonucleotide. Preferably, the nucleobasesequence of the modified oligonucleotide is 100% complementary to anucleobase sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, or SEQ ID NO: 237.

The antisense compound may comprise linked nucleosides. Preferably, theantisense compound is an antisense oligonucleotide.

The antisense compound may be a single-stranded or double-strandedoligonucleotide. Preferably, the antisense compound is a single-strandedoligonucleotide.

The antisense oligonucleotide may be modified, wherein at least oneinternucleoside linkage is a modified internucleoside linkage. Theinternucleoside linkage may be a phosphorothioate internucleosidelinkage.

The antisense oligonucleotide may be modified, wherein at least onenucleoside comprises a modified sugar. The modified sugar may be abicyclic sugar. Preferably, the at least one bicyclic sugar comprises a4′-CH(CH3)—O-2′ bridge. The modified sugar may comprise a2′-O-methoxyethyl. The antisense compound may comprise at least onetetrahydropyran modified nucleoside, wherein a tetrahydropyran ringreplaces the furanose ring. Preferably, each of the at least onetetrahydropyran modified nucleoside has the structure

wherein Bx is an optionally protected heterocyclic base moiety.

The antisense compound may comprise a modified nucleobase. The modifiednucleobase may be a 5-methylcytosine. Preferably, every cytosine is a5-methylcytosine.

The antisense compound may be a gapmer, for example an oligonucleotidecomprising:

a gap segment consisting of linked deoxynucleosides;

a 5′ wing segment consisting of linked nucleosides;

a 3′ wing segment consisting of linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment and wherein each nucleoside of each wing segmentcomprises a modified sugar. Preferably, each nucleoside of each wingsegment comprises a 2′-O-methoxyethyl sugar; and preferably eachinternucleoside linkage is a phosphorothioate linkage.

The antisense oligonucleotide may be a 5-10-5 MOE gapmer or a 2-15-3 MOEgapmer. The antisense oligonucleotide may consist of 20 linkednucleosides.

The antisense oligonucleotide may be a 4-10-4 MOE gapmer. The antisenseoligonucleotide may consist of 18 linked nucleosides.

Compositions described herein may comprise an oligonucleotide consistingof 12 to 30 linked nucleosides, targeted to a CD40 nucleic acid or asalt thereof and a pharmaceutically acceptable carrier or diluent.

The composition may comprise a single-stranded or double-strandedoligonucleotide.

Another embodiment of the invention is a pharmaceutical compositioncomprising an antisense compound as described above and a liposome or alipid based delivery system. Preferably, said liposome is an amphotericliposome. Preferably, said amphoteric liposome is formed from a lipidphase comprising an amphoteric lipid or a mixture of lipid componentswith amphoteric properties. Said amphoteric liposome may furthercomprise one or more neutral or zwitterionic lipids. More preferably,said amphoteric liposome is formed from a lipid phase comprising

-   -   (a) about 15 mol % POPC, about 45 mol % DOPE, about 20 mol %        MoChol, about 20 mol % Chems    -   (b) about 60 mol % POPC, about 10 mol % DOTAP, about 30 mol %        Chems    -   (c) about 30 mol % POPC, about 10 mol % DOTAP, about 20 mol %        Chems, about 40 mol % Chol    -   (d) about 60 mol % POPC, about 20 mol % HistChol, about 20 mol %        Chol.

A further embodiment of the invention is an antisense compound orcomposition as described above for medical use. Yet a further embodimentof the invention is an antisense compound or a composition as describedabove for the treatment of cancer or an inflammatory or immuneassociated condition. The treatment may further comprise administering asecond drug, which may be administered separately or concomitantly withthe antisense compound of the invention.

Methods described herein may comprise administering to an animal anantisense compound as described above, preferably an antisense compoundcomprising an oligonucleotide consisting of 12 to 30 linked nucleosidestargeted to a CD40 nucleic acid, or a composition comprising saidantisense compound. Preferably, the animal is a human.

Administration of the antisense compound and/or the second drug may beby parenteral administration, topical administration, oraladministration or aerosol administration. Parenteral administration maybe any of subcutaneous or intravenous administration.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

Definitions

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Where permitted, all patents, applications, publishedapplications and other publications, GENBANK Accession Numbers andassociated sequence information obtainable through databases such asNational Center for Biotechnology Information (NCBI) and other datareferred to throughout in the disclosure herein are incorporated byreference in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to anO-methoxy-ethyl modification of the 2′ position of a furosyl ring. A2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-O-methoxyethyl nucleotide” means a nucleotide comprising a2′-O-methoxyethyl modified sugar moiety.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5′ position. A 5-methylcytosine is a modifiednucleobase.

“Acceptable safety profile” means a pattern of side effects that iswithin clinically acceptable limits.

“Active pharmaceutical ingredient” means the substance or substances ina pharmaceutical composition that provides a desired effect.

“Active target region” means a target region to which one or more activeantisense compounds is targeted. “Active antisense compounds” meansantisense compounds that reduce target nucleic acid levels.

“Administered concomitantly” refers to the co-administration of twoagents in any manner in which the pharmacological effects of both aremanifest in the patient at the same time. Concomitant administrationdoes not require that both agents be administered in a singlepharmaceutical composition, in the same dosage form, or by the sameroute of administration.

“Administering” means providing a pharmaceutical agent to an individual,and includes, but is not limited to administering by a medicalprofessional and self-administering.

“Antisense compound” means an oligomeric compound that is capable ofundergoing hybridization to a target nucleic acid through hydrogenbonding.

“Antisense inhibition” means reduction of target nucleic acid levels inthe presence of an antisense compound complementary to a target nucleicacid compared to target nucleic acid levels in the absence of theantisense compound.

“Antisense oligonucleotide” means a single-stranded oligonucleotidehaving a nucleobase sequence that permits hybridization to acorresponding region or segment of a target nucleic acid.

“Bicyclic sugar” means a furosyl ring modified by the bridging of twonon-geminal ring atoms. A bicyclic sugar is a modified sugar.

“Bicyclic nucleic acid” or “BNA” or “bicyclic nucleoside” or “bicyclicnucleotide” refers to a nucleoside or nucleotide wherein the furanoseportion of the nucleoside includes a bridge connecting two carbon atomson the furanose ring, thereby forming a bicyclic ring system. As usedherein, unless otherwise indicated, the term “methyleneoxy BNA” alonerefers to β-D-methyleneoxy BNA.

“Cap structure” or “terminal cap moiety” means chemical modifications,which have been incorporated at either terminus of an antisensecompound.

“Chimeric antisense compounds” means antisense compounds that have atleast 2 chemically distinct regions, each position having a plurality ofsubunits. A “gapmer” means an antisense compound in which an internalposition having a plurality of nucleotides that supports RNaseH cleavageis positioned between external regions having one or more nucleotidesthat are chemically distinct from the nucleosides of the internalregion. A “gap segment” means the plurality of nucleotides that make upthe internal region of a gapmer. A “wing segment” means the externalregion of a gapmer.

“Co-administration” means administration of two or more pharmaceuticalagents to an individual. The two or more pharmaceutical agents may be ina single pharmaceutical composition, or may be in separatepharmaceutical compositions. Each of the two or more pharmaceuticalagents may be administered through the same or different routes ofadministration. Co-administration encompasses administration in parallelor sequentially.

“Complementarity” means the capacity for pairing between nucleobases ofa first nucleic acid and a second nucleic acid.

“Comply” means the adherence with a recommended therapy by a individual.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother.

“Diluent” means an ingredient in a composition that lackspharmacological activity, but is pharmaceutically necessary ordesirable. For example, in drugs that are injected the diluent may be aliquid, e.g. saline solution.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration, or in a specified time period. In certainembodiments, a dose may be administered in two or more boluses, tablets,or injections. For example, in certain embodiments, where subcutaneousadministration is desired, the desired dose requires a volume not easilyaccommodated by a single injection. In such embodiments, two or moreinjections may be used to achieve the desired dose. In certainembodiments, a dose may be administered in two or more injections tominimize injection site reaction in a individual. In other embodiments,the pharmaceutical agent is administered by infusion over an extendedperiod of time or continuously. Doses may be stated as the amount ofpharmaceutical agent per hour, day, week or month.

“Dosage unit” means a form in which a pharmaceutical agent is provided,e.g. pill, tablet, or other dosage unit known in the art. In certainembodiments, a dosage unit is a vial containing lyophilized antisenseoligonucleotide. In certain embodiments, a dosage unit is a vialcontaining reconstituted antisense oligonucleotide.

“Duration” means the period of time during which an activity or eventcontinues. In certain embodiments, the duration of treatment is theperiod of time during which doses of a pharmaceutical agent areadministered.

“Efficacy” means the ability to produce a desired effect.

“CD40 nucleic acid” means any nucleic acid encoding CD40. For example,in certain embodiments, a CD40 nucleic acid includes, withoutlimitation, a DNA sequence encoding CD40, an RNA sequence transcribedfrom DNA encoding CD40, and an mRNA sequence encoding CD40. “CD40 mRNA”means an mRNA encoding a CD40 protein.

“Fully complementary” means each nucleobase of a first nucleic acid hasa complementary nucleobase in a second nucleic acid. In certainembodiments, a first nucleic acid is an antisense compound and a targetnucleic acid is a second nucleic acid. In certain such embodiments, anantisense oligonucleotide is a first nucleic acid and a target nucleicacid is a second nucleic acid.

“Gap-widened” means an antisense compound has a gap segment of 12 ormore contiguous 2′-deoxyribonucleotides positioned between andimmediately adjacent to 5′ and 3′ wing segments having from one to sixnucleotides having modified sugar moieties. “Immediately adjacent” meansthere are no intervening nucleotides between the immediately adjacentelements.

“Hybridization” means the annealing of complementary nucleic acidmolecules. In certain embodiments, complementary nucleic acid moleculesinclude, but are not limited to, an antisense compound and a nucleicacid target. In certain such embodiments, complementary nucleic acidmolecules include, but are not limited to, an antisense oligonucleotideand a nucleic acid target

“Individual” means a human or non-human animal selected for treatment ortherapy.

“Individual compliance” means adherence to a recommended or prescribedtherapy by a individual.

“Injection site reaction” means inflammation or abnormal redness of skinat a site of injection in a individual.

“Internucleoside linkage” refers to the chemical bond betweennucleosides.

“Linked nucleosides” means adjacent nucleosides which are bondedtogether.

“Modified internucleoside linkage” refers to a substitution and/or anychange from a naturally occurring internucleoside bond (i.e. aphosphodiester internucleoside bond).

“Modified oligonucleotide” means an oligonucleotide comprising amodified internucleoside linkage, a modified sugar, and/or a modifiednucleobase.

“Modified sugar” refers to a substitution and/or any change from anatural sugar.

“Modified nucleobase” means any nucleobase other than adenine, cytosine,guanine, thymidine, or uracil. An “unmodified nucleobase” means thepurine bases adenine (A) and guanine (G), and the pyrimidine basesthymine (T), cytosine (C) and uracil (U).

“Modified nucleotide” means a nucleotide having, independently, amodified sugar moiety, modified internucleoside linkage, or modifiednucleobase. A “modified nucleoside” means a nucleotide having,independently, a modified sugar moiety or modified nucleobase.

“Modified sugar moiety” means a sugar moiety having any substitutionand/or change from a natural sugar moiety.

“Motif” means the pattern of unmodified and modified nucleosides in anantisense compound.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage.

“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA(2′-OH).

“Non-complementary nucleobase” or “mismatch” means a nucleobase of afirst nucleic acid that is not capable of pairing with the correspondingnucleobase of a second or target nucleic acid.

“Nucleoside” means a nucleobase linked to a sugar.

As used herein the term “nucleoside mimetic” is intended to includethose structures used to replace the sugar or the sugar and the base andnot necessarily the linkage at one or more positions of an oligomericcompound such as for example nucleoside mimetics having morpholino,cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugarmimetics e.g. non furanose sugar units.

“Nucleobase” means a heterocyclic moiety capable of pairing with a baseof another nucleic acid.

“Nucleobase sequence” means the order of contiguous nucleobasesindependent of any sugar, linkage, and/or nucleobase modification.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of the nucleoside.

The term “nucleotide mimetic” is intended to include those structuresused to replace the nucleoside and the linkage at one or more positionsof an oligomeric compound such as for example peptide nucleic acids ormorpholinos (morpholinos linked by —N(H)—C(═O)—O— or othernon-phosphodiester linkage).

“Oligomeric compound” means a polymer or oligomer of linked monomericsubunits which is capable of hybridizing to at least a region of anucleic acid molecule.

“Oligonucleotide” means an oligonucleotide in which the internucleosidelinkages do not contain a phosphorus atom.

“Oligonucleotide” means a polymer or oligomer of linked nucleosides eachof which can be modified or unmodified, independent one from another.

“Parenteral administration,” means administration through injection orinfusion. Parenteral administration includes, but is not limited to,subcutaneous administration, intravenous administration, orintramuscular administration.

“Pharmaceutical agent” means a substance that provides a therapeuticbenefit when administered to a individual. For example, in certainembodiments, an antisense oligonucleotide targeted to CD40 ispharmaceutical agent.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of antisense compounds, i.e., saltsthat retain the desired biological activity of the parentoligonucleotide and do not impart undesired toxicological effectsthereto.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual. For example, a pharmaceuticalcomposition may comprise one or more antisense oligonucleotides or acombination of antisense oligonucleotides and non-antisense activeagents and a sterile aqueous solution or other pharmaceuticallyacceptable additive.

“Phosphorothioate linkage” means a linkage between nucleosides where thephosphodiester bond is modified by replacing one of the non-bridgingoxygen atoms with a sulfur atom. A phosphorothioate linkage is amodified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e. linked) nucleobasesof a nucleic acid. In certain embodiments, a portion is a defined numberof contiguous nucleobases of a target nucleic acid. In certainembodiments, a portion is a defined number of contiguous nucleobases ofan antisense compound.

“Prodrug” means a therapeutic agent that is prepared in an inactive formthat is converted to an active form (i.e., drug) within the body orcells thereof by the action of endogenous enzymes or other chemicalsand/or conditions.

“Recommended therapy” means a therapeutic regimen recommended by amedical professional for the treatment, amelioration, or prevention of adisease.

“Side effects” means physiological responses attributable to a treatmentother than desired effects. In certain embodiments, side effectsinclude, without limitation, injection site reactions, liver functiontest abnormalities, renal function abnormalities, liver toxicity, renaltoxicity, central nervous system abnormalities, and myopathies. Forexample, increased aminotransferase levels in serum may indicate livertoxicity or liver function abnormality. For example, increased bilirubinmay indicate liver toxicity or liver function abnormality.

“Single-stranded modified oligonucleotide” means a modifiedoligonucleotide which is not hybridized to a complementary strand.

“Specifically hybridizable” means an antisense compound that hybridizesto a target nucleic acid to induce a desired effect, while exhibitingminimal or no effects on non-target nucleic acids.

The term “sugar surrogate” overlaps with the slightly broader term“nucleoside mimetic” but is intended to indicate replacement of thesugar unit (furanose ring) only. The tetrahydropyranyl rings providedherein are illustrative of an example of a sugar surrogate wherein thefuranose sugar group has been replaced with a tetrahydropyranyl ringsystem.

“Stringent hybridization conditions” means conditions under which anucleic acid molecule, such as an antisense compound, will hybridize toa target nucleic acid sequence, but to a minimal number of othersequences. Stringent conditions are sequence-dependent and will vary indifferent circumstances. In the context of this invention, “stringentconditions” under which oligomeric compounds hybridize to a targetsequence are determined by the nature and composition of the oligomericcompounds and the assays in which they are being investigated.

“Subcutaneous administration” means administration just below the skin.“Intravenous administration” means administration into a vein.

“Targeted” or “targeted to” means having a nucleobase sequence that willallow specific hybridization of an antisense compound to a targetnucleic acid to induce a desired effect. In certain embodiments, adesired effect is reduction of a target nucleic acid. In certain suchembodiments, a desired effect is reduction of a CD40 mRNA.

“Targeting” means the process of design and selection of an antisensecompound that will specifically hybridize to a target nucleic acid andinduce a desired effect.

“Target nucleic acid,” “target RNA,” “target RNA transcript” and“nucleic acid target” all mean a nucleic acid capable of being targetedby antisense compounds. Target nucleic acids may include, but are notlimited to, DNA, RNA (including, but not limited to pre-mRNA and mRNA orportions thereof) transcribed from DNA encoding a target, and alsomiRNA.

“Target region” means a portion of a target nucleic acid to which one ormore antisense compounds is targeted.

“Target segment” means the sequence of nucleotides of a target nucleicacid to which an antisense compound is targeted. “5′ target site” refersto the 5′-most nucleotide of a target segment. “3′ target site” refersto the 3′-most nucleotide of a target segment.

“Therapeutically effective amount” means an amount of a pharmaceuticalagent that provides a therapeutic benefit to an individual.

“Unmodified nucleotide” means a nucleotide composed of naturallyoccurring nucleobases, sugar moieties and internucleoside linkages. Incertain embodiments, an unmodified nucleotide is an RNA nucleotide(i.e., β-D-ribonucleosides) or a DNA nucleotide (i.e.,β-D-deoxyribonucleoside).

Antisense Compounds

Antisense compounds include, but are not limited to, oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics,antisense oligonucleotides, and siRNAs. An oligomeric compound may be“antisense” to a target nucleic acid, meaning that is capable ofundergoing hybridization to a target nucleic acid through hydrogenbonding.

In certain embodiments, an antisense compound has a nucleobase sequencethat, when written in the 5′ to 3′ direction, comprises the reversecomplement of the target segment of a target nucleic acid to which it istargeted. In certain such embodiments, an antisense oligonucleotide hasa nucleobase sequence that, when written in the 5′ to 3′ direction,comprises the reverse complement of the target segment of a targetnucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to a CD40 nucleicacid is 12 to 30 subunits in length. In other words, antisense compoundsare from 12 to 30 linked subunits. In other embodiments, the antisensecompound is 8 to 80, 12 to 50, 15 to 30, 18 to 24, 19 to 22, or 20linked subunits. In certain such embodiments, the antisense compoundsare 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, or 80 linked subunits in length, or a range defined by any two ofthe above values. In some embodiments the antisense compound is anantisense oligonucleotide, and the linked subunits are nucleotides.

In certain embodiments, a shortened or truncated antisense compoundtargeted to a CD40 nucleic acid has a single subunit deleted from the 5′end (5′ truncation), or alternatively from the 3′ end (3′ truncation). Ashortened or truncated antisense compound targeted to a CD40 nucleicacid may have two subunits deleted from the 5′ end, or alternatively mayhave two subunits deleted from the 3′ end, of the antisense compound.Alternatively, the deleted nucleosides may be dispersed throughout theantisense compound, for example, in an antisense compound having onenucleoside deleted from the 5′ end and one nucleoside deleted from the3′ end.

When a single additional subunit is present in a lengthened antisensecompound, the additional subunit may be located at the 5′ or 3′ end ofthe antisense compound. When two are more additional subunits arepresent, the added subunits may be adjacent to each other, for example,in an antisense compound having two subunits added to the 5′ end (5′addition), or alternatively to the 3′ end (3′ addition), of theantisense compound. Alternatively, the added subunits may be dispersedthroughout the antisense compound, for example, in an antisense compoundhaving one subunit added to the 5′ end and one subunit added to the 3′end.

It is possible to increase or decrease the length of an antisensecompound, such as an antisense oligonucleotide, and/or introducemismatch bases without eliminating activity. For example, in Woolf etal. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series ofantisense oligonucleotides 13-25 nucleobases in length were tested fortheir ability to induce cleavage of a target RNA in an oocyte injectionmodel. Antisense oligonucleotides 25 nucleobases in length with 8 or 11mismatch bases near the ends of the antisense oligonucleotides were ableto direct specific cleavage of the target mRNA, albeit to a lesserextent than the antisense oligonucleotides that contained no mismatches.Similarly, target specific cleavage was achieved using 13 nucleobaseantisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001)demonstrated the ability of an oligonucleotide having 100%complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xLmRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and invivo. Furthermore, this oligonucleotide demonstrated potent anti-tumoractivity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a seriesof tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42nucleobase antisense oligonucleotides comprised of the sequence of twoor three of the tandem antisense oligonucleotides, respectively, fortheir ability to arrest translation of human DHFR in a rabbitreticulocyte assay. Each of the three 14 nucleobase antisenseoligonucleotides alone was able to inhibit translation, albeit at a moremodest level than the 28 or 42 nucleobase antisense oligonucleotides.

Bhanot et al. (PCT/US2007/068401) provided short antisense compounds,including compounds comprising chemically-modified high-affinitymonomers 8 to 16 monomers in length. These short antisense compoundswere shown to be useful for reducing target nucleic acids and/orproteins in cells, tissues, and animals with increased potency andimproved therapeutic index. Short antisense compounds were effective atlower doses than previously described antisense compounds, allowing fora reduction in toxicity and cost of treatment. In addition, thedescribed short antisense compounds have greater potential for oraldosing.

Antisense Compound Motifs

In certain embodiments, antisense compounds targeted to a CD40 nucleicacid have chemically modified subunits arranged in patterns, or motifs,to confer to the antisense compounds properties such as enhanced theinhibitory activity, increased binding affinity for a target nucleicacid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, increased binding affinity for the targetnucleic acid, and/or increased inhibitory activity. A second region of achimeric antisense compound may optionally serve as a substrate for thecellular endonuclease RNase H, which cleaves the RNA strand of anRNA:DNA duplex.

Antisense compounds having a gapmer motif are considered chimericantisense compounds. In a gapmer an internal region having a pluralityof nucleotides that supports RNaseH cleavage is positioned betweenexternal regions having a plurality of nucleotides that are chemicallydistinct from the nucleosides of the internal region. In the case of anantisense oligonucleotide having a gapmer motif, the gap segmentgenerally serves as the substrate for endonuclease cleavage, while thewing segments comprise modified nucleosides. In a preferred embodiment,the regions of a gapmer are differentiated by the types of sugarmoieties comprising each distinct region. The types of sugar moietiesthat are used to differentiate the regions of a gapmer may in someembodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides,2′-modified nucleosides (such 2′-modified nucleosides may include2′-MOE, and 2′-O—CH₃, among others), and bicyclic sugar modifiednucleosides (such bicyclic sugar modified nucleosides may include thosehaving a 4′-(CH₂)n-O-2′ bridge, where n=1 or n=2). Preferably, eachdistinct region comprises uniform sugar moieties. The wing-gap-wingmotif is frequently described as “X-Y-Z”, where “X” represents thelength of the 5′ wing region, “Y” represents the length of the gapregion, and “Z” represents the length of the 3′ wing region. Any of theantisense compounds described herein can have a gapmer motif. In someembodiments, X and Z are the same, in other embodiments they aredifferent. In a preferred embodiment, Y is between 8 and 15 nucleotides.X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides. Thus, gapmers of thepresent invention include, but are not limited to, for example 5-10-5,4-8-4, 4-10-4, 2-15-3, 4-12-3, 4-12-4, 3-14-3, 2-16-2, 1-18-1, 3-10-3,2-10-2, 1-10-1 or 2-8-2.

In some embodiments, the antisense compound as a “wingmer” motif, havinga wing-gap or gap-wing configuration, i.e. an X-Y or Y-Z configurationas described above for the gapmer configuration. Thus, wingmerconfigurations of the present invention include, but are not limited to,for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10 or8-2.

In one embodiment, antisense compounds targeted to a CD40 nucleic acidpossess a 5-10-5 gapmer motif.

In some embodiments, an antisense compound targeted to a CD40 nucleicacid has a gap-widened motif. In other embodiments, an antisenseoligonucleotide targeted to a CD40 nucleic acid has a gap-widened motif.

In one embodiment, a gap-widened antisense oligonucleotide targeted to aCD40 nucleic acid has a gap segment of fourteen 2′-deoxyribonucleotidespositioned between wing segments of three chemically modifiednucleosides. In one embodiment, the chemical modification comprises a2′-sugar modification. In another embodiment, the chemical modificationcomprises a 2′-MOE sugar modification.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode CD40 include, without limitation, thefollowing: GENBANK Accession No. X60592.1, first deposited with GENBANK®on Apr. 21, 1993 and incorporated herein as SEQ ID NO: 1; GENBANK®Accession No. H50598.1, first deposited with GENBANK® on Sep. 19, 1995,and incorporated herein as SEQ ID NO: 2; GENBANK Accession No.AA203290.1, first deposited with GENBANK® on Jan. 25, 1997, andincorporated herein as SEQ ID NO: 3; and nucleotides 9797000 to 9813000of GENBANK Accession No. NT_011362.9, first deposited with GENBANK® onNov. 29, 2000, and incorporated herein as SEQ ID NO: 4, and GENBANK®Accession No. BC064518.1, incorporated herein as SEQ ID NO: 237.

It is understood that the sequence set forth in each SEQ ID NO in theExamples contained herein is independent of any modification to a sugarmoiety, an internucleoside linkage, or a nucleobase. As such, antisensecompounds defined by a SEQ ID NO may comprise, independently, one ormore modifications to a sugar moiety, an internucleoside linkage, or anucleobase. Antisense compounds described by Isis Number (Isis No)indicate a combination of nucleobase sequence and motif.

In one embodiment, a target region is a structurally defined region ofthe nucleic acid. For example, a target region may encompass a 3′ UTR, a5′ UTR, an exon, an intron, a coding region, a translation initiationregion, translation termination region, or other defined nucleic acidregion. The structurally defined regions for CD40 can be obtained byaccession number from sequence databases such as NCBI and suchinformation is incorporated herein by reference. In other embodiments, atarget region may encompass the sequence from a 5′ target site of onetarget segment within the target region to a 3′ target site of anothertarget segment within the target region.

Targeting includes determination of at least one target segment to whichan antisense compound hybridizes, such that a desired effect occurs. Incertain embodiments, the desired effect is a reduction in mRNA targetnucleic acid levels. In other embodiments, the desired effect isreduction of levels of protein encoded by the target nucleic acid or aphenotypic change associated with the target nucleic acid.

A target region may contain one or more target segments. Multiple targetsegments within a target region may be overlapping. Alternatively, theymay be non-overlapping. In one embodiment, target segments within atarget region are separated by no more than about 300 nucleotides. Inother embodiments, target segments within a target region are separatedby no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30,20, or 10 nucleotides on the target nucleic acid. In another embodiment,target segments within a target region are separated by no more thanabout 5 nucleotides on the target nucleic acid. In additionalembodiments, target segments are contiguous.

Suitable target segments may be found within a 5′ UTR, a coding region,a 3′ UTR, an intron, or an exon. Target segments containing a startcodon or a stop codon are also suitable target segments. A suitabletarget segment may specifically exclude a certain structurally definedregion such as the start codon or stop codon.

The determination of suitable target segments may include a comparisonof the sequence of a target nucleic acid to other sequences throughoutthe genome. For example, the BLAST algorithm may be used to identifyregions of similarity amongst different nucleic acids. This comparisoncan prevent the selection of antisense compound sequences that mayhybridize in a non-specific manner to sequences other than a selectedtarget nucleic acid (i.e., non-target or off-target sequences).

There may be variation in activity (e.g., as defined by percentreduction of target nucleic acid levels) of the antisense compoundswithin an active target region. In one embodiment, reductions in CD40mRNA levels are indicative of inhibition of CD40 expression. Reductionsin levels of a CD40 protein are also indicative of inhibition of targetmRNA expression. Further, phenotypic changes are indicative ofinhibition of CD40 expression. For example, changes in cell morphologyover time or treatment dose as well as changes in levels of cellularcomponents such as proteins, lipids, nucleic acids, hormones,saccharides, or metals is indicative of inhibition of CD40 expression.Reduction of eosinophils is indicative of inhibition of CD40 expression.Measurements of cellular status which include pH, stage of cell cycle,intake or excretion of biological indicators by the cell are alsoendpoints of interest.

Analysis of the genotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the CD40 inhibitors. Hallmarkgenes, or those genes suspected to be associated with a specific diseasestate, condition, or phenotype, are measured in both treated anduntreated cells.

Genomic Structure, Exons and Introns

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also preferred target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesare known as “fusion transcripts”. It is also known that introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

Hybridization

In some embodiments, hybridization occurs between an antisense compounddisclosed herein and a CD40 nucleic acid. The most common mechanism ofhybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteenor reversed Hoogsteen hydrogen bonding) between complementarynucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditionsare sequence-dependent and are determined by the nature and compositionof the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizableto a target nucleic acid are well known in the art. In one embodiment,the antisense compounds provided herein are specifically hybridizablewith a CD40 nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary toeach other when a sufficient number of nucleobases of the antisensecompound can hydrogen bond with the corresponding nucleobases of thetarget nucleic acid, such that a desired effect will occur (e.g.,antisense inhibition of a target nucleic acid, such as a CD40 nucleicacid).

Non-complementary nucleobases between an antisense compound and a CD40nucleic acid may be tolerated provided that the antisense compoundremains able to specifically hybridize to a target nucleic acid.Moreover, an antisense compound may hybridize over one or more segmentsof a CD40 nucleic acid such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure,mismatch or hairpin structure).

In some embodiments, the antisense compounds provided herein are atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%complementary to a CD40 nucleic acid. Percent complementarity of anantisense compound with a target nucleic acid can be determined usingroutine methods. For example, an antisense compound in which 18 of 20nucleobases of the antisense compound are complementary to a targetregion, and would therefore specifically hybridize, would represent 90percent complementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden,Genome Res., 1997, 7, 649 656). Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., 1981, 2, 482 489).

In other embodiments, the antisense compounds provided herein are fullycomplementary (i.e, 100% complementary) to a target nucleic acid. Forexample, an antisense compound may be fully complementary to a CD40nucleic acid, or a target region, or a target segment or target sequencethereof. As used herein, “fully complementary” means each nucleobase ofan antisense compound is capable of precise base pairing with thecorresponding nucleobases of a target nucleic acid.

The location of a non-complementary nucleobase may be at the 5′ end or3′ end of the antisense compound. Alternatively, the non-complementarynucleobase or nucleobases may be at an internal position of theantisense compound. When two or more non-complementary nucleobases arepresent, they may be contiguous (i.e. linked) or non-contiguous. In oneembodiment, a non-complementary nucleobase is located in the wingsegment of a gapmer antisense oligonucleotide.

In one embodiment, antisense compounds up to 20 nucleobases in lengthcomprise no more than 4, no more than 3, no more than 2 or no more than1 non-complementary nucleobase(s) relative to a target nucleic acid,such as a CD40 nucleic acid.

In another embodiment, antisense compounds up to 30 nucleobases inlength comprise no more than 6, no more than 5, no more than 4, no morethan 3, no more than 2 or no more than 1 non-complementary nucleobase(s)relative to a target nucleic acid, such as a CD40 nucleic acid.

The antisense compounds provided herein also include those which arecomplementary to a portion of a target nucleic acid. As used herein,“portion” refers to a defined number of contiguous (i.e. linked)nucleobases within a region or segment of a target nucleic acid. A“portion” can also refer to a defined number of contiguous nucleobasesof an antisense compound. In one embodiment, the antisense compounds arecomplementary to at least an 8 nucleobase portion of a target segment.In another embodiment, the antisense compounds are complementary to atleast a 12 nucleobase portion of a target segment. In yet anotherembodiment, the antisense compounds are complementary to at least a 15nucleobase portion of a target segment. Also contemplated are antisensecompounds that are complementary to at least a 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment,or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percentidentity to a particular nucleotide sequence, SEQ ID NO, or compoundrepresented by a specific Isis number. As used herein, an antisensecompound is identical to the sequence disclosed herein if it has thesame nucleobase pairing ability. For example, a RNA which containsuracil in place of thymidine in a disclosed DNA sequence would beconsidered identical to the DNA sequence since both uracil and thymidinepair with adenine. Shortened and lengthened versions of the antisensecompounds described herein as well as compounds having non-identicalbases relative to the antisense compounds provided herein also arecontemplated. The non-identical bases may be adjacent to each other ordispersed throughout the antisense compound. Percent identity of anantisense compound is calculated according to the number of bases thathave identical base pairing relative to the sequence to which it isbeing compared.

In one embodiment, the antisense compounds are at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more ofthe antisense compounds or SEQ ID NOs, or a portion thereof, disclosedherein.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known asbase) portion of the nucleoside is normally a heterocyclic base moiety.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.Oligonucleotides are formed through the covalent linkage of adjacentnucleosides to one another, to form a linear polymeric oligonucleotide.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide.

Modifications to antisense compounds encompass substitutions or changesto internucleoside linkages, sugar moieties, or nucleobases. Modifiedantisense compounds are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for nucleic acid target, increased stability in thepresence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase thebinding affinity of a shortened or truncated antisense oligonucleotidefor its target nucleic acid. Consequently, comparable results can oftenbe obtained with shorter antisense compounds that have such chemicallymodified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′to 5′ phosphodiester linkage. Antisense compounds having one or moremodified, i.e. non-naturally occurring, internucleoside linkages areoften selected over antisense compounds having naturally occurringinternucleoside linkages because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for target nucleicacids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages includeinternucleoside linkages that retain a phosphorus atom as well asinternucleoside linkages that do not have a phosphorus atom.Representative phosphorus containing internucleoside linkages include,but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphonoacetates, phosphoramidate, andphosphorothioates or phosphorodithioates. Internucleoside linkages thatdo not have a phosphorus atom include, amongst others,methylene(methylimino) or MMI linkages, morpholino linkages or amidelinkages. In peptide nucleic acids (PNA) the sugar backbone is replacedwith an amide containing backbone.

Methods of preparation of phosphorous-containing andnon-phosphorous-containing linkages are well known.

Representative United States patents that teach the preparation ofphosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697;5,625,050 and U.S. Pat. No. 6,693,187, each of which is hereinincorporated by reference.

Representative United States patents that teach the preparation ofnon-phosphorous-containing linkages include, but are not limited to,U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677;5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439,each of which is herein incorporated by reference.

In one embodiment, antisense compounds targeted to a CD40 nucleic acidcomprise one or more modified internucleoside linkages. In someembodiments, the modified internucleoside linkages are phosphorothioatelinkages. In other embodiments, each internucleoside linkage of anantisense compound is a phosphorothioate internucleoside linkage.

Modified Sugar Moieties

Antisense compounds of the invention can optionally contain one or morenucleosides wherein the sugar group has been modified. Such sugarmodified nucleosides may impart enhanced nuclease stability, increasedbinding affinity or some other beneficial biological property to theantisense compounds. In certain embodiments, nucleosides are modified bymodification of the ribofuranose ring. Such modifications includewithout limitation, addition of substituent groups, bridging ofnon-geminal ring atoms to form a bicyclic nucleic acid (BNA), as inlocked nucleic acids (LNA), replacement of the ribosyl ring oxygen atomwith S, N(R), or C(R1)(R)2 (R═H, C1-C12 alkyl or a protecting group) andcombinations thereof. Examples of chemically modified sugars include2′-F-5′-methyl substituted nucleoside (see PCT International ApplicationWO 2008/101157 published on Aug. 21, 2008 for other disclosed 5′,2′-bissubstituted nucleosides) or replacement of the ribosyl ring oxygen atomwith S with further substitution at the 2′-position (see published U.S.Patent Application US2005-0130923, published on Jun. 16, 2005) oralternatively 5′-substitution of a BNA (see PCT InternationalApplication WO 2007/134181 Published on Nov. 22, 2007 wherein LNA issubstituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include withoutlimitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S,2′-F, 2′-OCH₃ (known as 2′-OMe) and 2′-O(CH₂)₂OCH₃ (known as 2′MOE)substituent groups. The substituent at the 2′ position can also beselected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, OCF₃,O—CH₂CH₂CH₂NH₂, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(Rm)(Rn) such as2′-dimethylaminooxyethoxy (2′-O—(CH₂)₂ON(CH₃)₂ or 2′-DMAOE),O(CH₂)₂—O—(CH₂)₂—N(Rm)(Rn) such as 2′-dimethylaminoethoxyethoxy(2′-O—(CH₂)₂—O—(CH₂)₂—N(CH₃)₂ or 2′-DMAEOE and O—CH₂—C(═O)—N(Rm)(Rn),where each Rm and Rn is, independently, H or substituted orunsubstituted C1-C10 alkyl.

Examples of bicyclic nucleic acids (BNAs) include without limitationnucleosides comprising a bridge between the 4′ and the 2′ ribosyl ringatoms, e.g. a 4′-(CH₂)_(n)—O-2′ bridge, where n=1 or n=2. In certainembodiments, antisense compounds provided herein include one or more BNAnucleosides wherein the bridge comprises one of the formulas:4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)₂—O-2′(ENA); 4′-C(CH₃)₂—O-2′ (see PCT/US2008/068922); 4′-CH(CH₃)—O-2′ and4′CH(CH₂OCH₃)—O-2′ (see U.S. Pat. No. 7,399,845, issued on Jul. 15,2008); 4′-CH₂—N(OCH₃)-2′ (see PCT/US2008/064591); 4′-CH₂—O—N(CH₃)-2′(see published U.S. Patent Application US2004-0171570, published Sep. 2,2004); 4′-CH₂—N(R)—O-2′ (see U.S. Pat. No. 7,427,672, issued on Sep. 23,2008); 4′-CH₂—C(CH₃)-2′ and 4′-CH₂—C(═CH₂)-2′ (see PCT/US2008/066154);and wherein R is, independently, H, C1-C12 alkyl, or a protecting group.Each of the foregoing BNAs include various stereochemical sugarconfigurations including for example α-L-ribofuranose andβ-D-ribofuranose (See PCT international application PCT/DK98/00393,published on Mar. 25, 1999 as WO99/14226).

In certain embodiments, nucleosides are modified by replacement of theribosyl ring with a sugar surrogate. Such modification includes withoutlimitation, replacement of the ribosyl ring with a surrogate ring system(sometimes referred to as DNA analogs) such as a morpholino ring, acyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring such asone having one of the formula:

wherein Bx is an optionally protected heterocyclic base moiety.

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknow in the art that can be used to modify nucleosides for incorporationinto antisense compounds (see for example Leumann, C. J., Bioorg. Med.Chem. 10, (2002), 841-854). Such ring systems can undergo variousadditional substitutions to enhance activity.

Methods for the preparations of modified sugars are well known to thoseskilled in the art. Representative United States patents that teach thepreparation of modified sugars include, but are not limited to U.S. Pat.No. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; and 5,700,920, each of which is herein incorporated byreference.

In certain embodiments, a 2′-modified nucleoside has a bicyclic sugarmoiety. In certain such embodiments, the bicyclic sugar moiety is a Dsugar in the alpha configuration. In certain such embodiments, thebicyclic sugar moiety is a D sugar in the beta configuration. In certainsuch embodiments, the bicyclic sugar moiety is an L sugar in the alphaconfiguration. In certain such embodiments, the bicyclic sugar moiety isan L sugar in the beta configuration.

In certain embodiments, the bicyclic sugar moiety comprises a bridgegroup between the 2′ and the 4′-carbon atoms. In certain suchembodiments, the bridge group comprises from 1 to 8 linked biradicalgroups. In certain embodiments, the bicyclic sugar moiety comprises from1 to 4 linked biradical groups. In certain embodiments, the bicyclicsugar moiety comprises 2 or 3 linked biradical groups. In certainembodiments, the bicyclic sugar moiety comprises 2 linked biradicalgroups. In certain embodiments, a linked biradical group is selectedfrom —O—, —S—, —N(R1)-, —C(R1)(R2)-, —C(R1)=C(R1)-, —C(R1)=N—,—C(═NR1)-, —Si(R1)(R2)-, —S(═O)₂—, —S(═O)—, —C(═O)— and —C(═S)—; whereeach R1 and R2 is, independently, H, hydroxyl, C1-C12 alkyl, substitutedC1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20aryl, a heterocycle radical, a substituted hetero-cycle radical,heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substitutedC5-C7 alicyclic radical, halogen, substituted oxy (—O—), amino,substituted amino, azido, carboxyl, substituted carboxyl, acyl,substituted acyl, CN, thiol, substituted thiol, sulfonyl (S(═O)₂—H),substituted sulfonyl, sulfoxyl (S(═O)—H) or substituted sulfoxyl; andeach substituent group is, independently, halogen, C1-C12 alkyl,substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl,C2-C12 alkynyl, substituted C2-C12 alkynyl, amino, substituted amino,acyl, substituted acyl, C1-C12 aminoalkyl, C1-C12 aminoalkoxy,substituted C1-C12 aminoalkyl, substituted C1-C12 aminoalkoxy or aprotecting group.

In some embodiments, the bicyclic sugar moiety is bridged between the 2′and 4′ carbon atoms with a biradical group selected from —O—(CH₂)p-,—O—CH₂—, —O—CH₂CH2, —O—CH(alkyl)-, —NH—(CH₂)p-, —N(alkyl)-(CH₂)p-,—O—CH(alkyl)-, —(CH(alkyl))(CH₂)p-, —NH—O—(CH₂)p-, —N(alkyl)-O—(CH₂)p-,or —O—N(alkyl)-(CH₂)p-, wherein p is 1, 2, 3, 4 or 5 and each alkylgroup can be further substituted. In certain embodiments, p is 1, 2 or3.

In one aspect, each of said bridges is, independently, —[C(R1)(R2)]n-,—[C(R1)(R2)]n-O—, —C(R1R2)-N(R1)-O— or —C(R1R2)-O—N(R1)-. In anotheraspect, each of said bridges is, independently,4′-(CH₂)₃-2′,4′-(CH₂)₂-2′,4′-CH₂—O-2′,4′-(CH₂)₂—O-2′,4′-CH₂—O—N(R1)-2′and 4′-CH₂—N(R1)-O-2′- wherein each R1 is, independently, H, aprotecting group or C1-C12 alkyl.

In nucleotides having modified sugar moieties, the nucleobase moieties(natural, modified or a combination thereof) are maintained forhybridization with an appropriate nucleic acid target.

In one embodiment, antisense compounds targeted to a CD40 nucleic acidcomprise one or more nucleotides having modified sugar moieties. In apreferred embodiment, the modified sugar moiety is 2′-MOE. In otherembodiments, the 2′-MOE modified nucleotides are arranged in a gapmermotif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic unmodified nucleobases. Both natural and modifiednucleobases are capable of participating in hydrogen bonding. Suchnucleobase modifications may impart nuclease stability, binding affinityor some other beneficial biological property to antisense compounds.Modified nucleobases include synthetic and natural nucleobases such as,for example, 5-methylcytosine (5-me-C). Certain nucleobasesubstitutions, including 5-methylcytosine substitutions, areparticularly useful for increasing the binding affinity of an antisensecompound for a target nucleic acid. For example, 5-methylcytosinesubstitutions have been shown to increase nucleic acid duplex stabilityby 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds.,Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.276-278).

Additional unmodified nucleobases include 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Nucleobases that are particularly useful for increasing the bindingaffinity of antisense compounds include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

Methods for the preparations of modified nucleobases are well known tothose skilled in the art. Representative United States patents thatteach the preparation of modified nucleobases include, but are notlimited to U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205;5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588;6,005,096; 5,750,692 and 5,681,941 each of which is herein incorporatedby reference.

In one embodiment, antisense compounds targeted to a CD40 nucleic acidcomprise one or more modified nucleobases. In an additional embodiment,gap-widened antisense oligonucleotides targeted to a CD40 nucleic acidcomprise one or more modified nucleobases. In some embodiments, themodified nucleobase is 5-methylcytosine. In further embodiments, eachcytosine is a 5-methylcytosine.

Conjugated Antisense Compounds

Antisense compounds may be covalently linked to one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the resulting antisense oligonucleotides. Typical conjugategroups include cholesterol moieties and lipid moieties. Additionalconjugate groups include carbohydrates, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizinggroups that are generally attached to one or both termini of antisensecompounds to enhance properties such as, for example, nucleasestability. Included in stabilizing groups are cap structures. Theseterminal modifications protect the antisense compound having terminalnucleic acid from exonuclease degradation, and can help in deliveryand/or localization within a cell. The cap can be present at the5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be presenton both termini. Cap structures are well known in the art and include,for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizinggroups that can be used to cap one or both ends of an antisense compoundto impart nuclease stability include those disclosed in WO 03/004602published on Jan. 16, 2003.

Compositions and Methods for Formulating Pharmaceutical Compositions

Antisense oligonucleotides may be admixed with pharmaceuticallyacceptable active and/or inert substances for the preparation ofpharmaceutical compositions or formulations. Compositions and methodsfor the formulation of pharmaceutical compositions are dependent upon anumber of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Antisense compound targeted to a CD40 nucleic acid can be utilized inpharmaceutical compositions by combining the antisense compound with asuitable pharmaceutically acceptable diluent or carrier. Apharmaceutically acceptable diluent includes for examplephosphate-buffered saline (PBS). PBS is a diluent suitable for use incompositions to be delivered parenterally. Accordingly, in oneembodiment, employed in the methods described herein is a pharmaceuticalcomposition comprising an antisense compound targeted to a CD40 nucleicacid and a pharmaceutically acceptable diluent. In one embodiment, thepharmaceutically acceptable diluent is PBS. In other embodiments, theantisense compound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other oligonucleotide which, upon administration to an animal,including a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof. Accordingly, forexample, the disclosure is also drawn to pharmaceutically acceptablesalts of antisense compounds, prodrugs, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents. Suitablepharmaceutically acceptable salts include, but are not limited to,sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an antisense compound which are cleaved by endogenousnucleases within the body, to form the active antisense compound.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug which may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes which are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety.

Other liposomes or lipid based delivery systems known in the art aredescribed for example in WO 05/105152; WO 06/069782; Morrissey et al.,Nature Biotechnology, 23 (8), 1002-1007, 2005; WO 05/007196; Wheeler etal., Gene Therapy, 6 (2), 271-281, 1999; WO 02/34236; Budker et al.,Nature Biotechnology, 14 (6), 760-764, 1996; U.S. Pat. No. 5,965,434;U.S. Pat. No. 5,635,487; Spagnou et al., Biochemistry, 43 (42),13348-13356, 2004; U.S. Pat. No. 6,756,054; WO 06/016097 and U.S. Pat.No. 5,785,992; WO 04/035523, each of which is herein incorporated byreference.

In a preferred embodiment of the invention amphoteric liposomes may beused as formulations which include the inventive antisense compounds.Amphoteric liposomes are a class of liposomes having an anionic orneutral charge at pH 7.5 and a cationic charge at pH 4. Reference ismade to WO 02/066012 by Panzner et al. which is incorporated herein byreference. The use, selection and manufacturing of amphoteric liposomesfor the transfection of cells is further described in WO 05/094783 ofEnded et al., WO 07/031,333 of Panzner et al., WO 07/107,304 of Panzneret al. and WO 08/043,575 of Panzner et al. Amphoteric liposomes have anexcellent bio-distribution and are well tolerated in animals. They canencapsulate nucleic acid molecules with high efficiency. WO 06/048329 ofPanzner et al., which is incorporated herein by reference in itsentirety, describes pharmaceutical compositions comprising amphotericliposomes and oligonucleotides which are adapted to target nucleic acidsencoding CD40.

By “amphoteric” is meant herein that the liposomes comprise chargedgroups of both anionic and cationic character wherein:

(i) at least one of the charged groups has a pKa between 4 and 7.4,

(ii) the cationic charge prevails at pH 4 and

(iii) the anionic charge prevails at pH 7.4;

whereby the liposomes have an isoelectric point of zero net chargebetween pH 4 and pH 7.4. Amphoteric character, by this definition, isdifferent from “zwitterionic character”, because zwitterions do not havea pK in the range mentioned above. In consequence, zwitterions areessentially neutral over a range of pH values. Phosphatidylcholine orphosphatidylethanolamines, for example, are neutral lipids withzwitterionic character.

Amphoteric liposomes may be formed from a lipid phase comprising anamphoteric lipid. In some embodiments said lipid phase may comprise 5 to30 mol. % of said amphoteric lipid, preferably 10 to 25 mol. %.

Suitable amphoteric lipids are disclosed in WO 02/066489 and WO03/070735 by Panzner et al. Preferably, said amphoteric lipid isselected from the group consisting of HistChol, HistDG, isoHistSuccDG,Acylcarnosin and HCChol. (A glossary of such abbreviated forms of thenames of the lipids referred to herein is included below for ease ofreference. A number of such abbreviations are those that are commonlyused by those skilled in the art.)

Alternatively, said amphoteric liposomes may be formed from a lipidphase comprising a mixture of lipid components with amphotericproperties. Such amphoteric liposomes may be formed from pH-responsiveanionic and/or cationic components, as disclosed for example in WO02/066012. Cationic lipids sensitive to pH are disclosed in WO 02/066489and WO 03/070220 and in the references made therein, in particular inBudker, et al. 1996, Nat Biotechnol. 14 (6):760-4, and can be used incombination with constitutively charged anionic lipids or with anioniclipids that are sensitive to pH.

Alternatively, the cationic charge may be introduced from constitutivelycharged lipids that are known to those skilled in the art in combinationwith a pH sensitive anionic lipid. Combinations of constitutivelycharged anionic and cationic lipids, e.g. DOTAP and DPPG, are notpreferred. Thus, in some presently preferred embodiments of theinvention, said mixture of lipid components may comprise (i) a stablecationic lipid and a chargeable anionic lipid, (ii) a chargeablecationic lipid and chargeable anionic lipid or (iii) a stable anioniclipid and a chargeable cationic lipid.

Preferred cationic components include DMTAP, DPTAP, DOTAP, DC-Chol,MoChol, HisChol, DPIM, CHIM, DOME, DDAB, DAC-Chol, TC-Chol, DOTMA, DOGS,(C18)₂Gly⁺ N,N-dioctadecylamido-glycin, CTAB, CPyC, DODAP and DOEPC.

Preferred anionic lipids for use with the invention include DOGSucc,POGSucc, DMGSucc, DPGSucc, DMPS, DPPS, DOPS, POPS, DMPG, DPPG, DOPG,POPG, DMPA, DPPA, DOPA, POPA, CHEMS and CetylP.

Preferably, such an amphoteric mixture of lipids does not constitutemore than about 70 mol. % of the lipid phase. In some embodiments, saidmixture may constitute not more than 50 mol. % of the lipid phase;preferably said lipid phase comprises about 20 to about 40 mol. % ofsuch a mixture.

In some embodiments, said lipid phase may further comprise a neutrallipid, preferably a neutral phospholipid, such as a phosphatidylcholine.Presently preferred phosphatidylcholines include POPC, natural orhydrogenated soy bean PC, natural or hydrogenated egg PC, DMPC, DPPC,DSPC and DOPC. More preferably, said phosphatidylcholine comprises POPC,non-hydrogenated soy bean PC or non-hydrogenated egg PC.

The lipid phase may comprise at least 15 mol. % of saidphosphatidylcholine, preferably at least 20 mol. %. In some embodiments,said lipid phase may comprise no less than about 25 mol. %phosphatidylcholine. Alternatively, said lipid phase may comprise noless than about 40 mol. % phosphatidylcholine.

A presently preferred formulation in accordance with the presentinvention comprises a liposome having about 60 mol. % POPC, about 10mol. % DOTAP and about 30 mol. % CHEMS.

In some embodiments said neutral lipid may comprise aphosphatidylethanolamine or a mixture of phosphatidylcholine andphosphatidylethanolamine. Said neutral phosphatidylcholines orphosphatidylethanolamines or mixtures of the two may be present in thelipid phase in the molar amount (mol. %) not constituted by the othercomponents of the lipid phase, but to at least 20 mol. % (the total forthe lipid phase being 100 mol. %).

Preferred phosphatidylethanolamines include DOPE, DMPE and DPPE.

In some embodiments said neutral lipid may comprise POPC and DOPE.

Advantageously, said lipid phase may comprise a mixture of anionic andcationic lipids with amphoteric properties, phosphatidylcholine andphosphatidylethanolamine. Amphoteric liposomes formed from such a lipidphase may be serum-stable and therefore suitable for systemic delivery.Preferably said lipid phase comprises MoChol as a cationic lipid andCHEMS or DMG-Succ as an anionic lipid.

Further presently preferred amphoteric liposomes for use as formulationswhich include antisense compounds of the present invention may beselected from the group consisting of:

-   -   (a) about 15 mol. % POPC, about 45 mol. % DOPE, about 20 mol. %        MoChol and about 20 mol. % CHEMS;    -   (b) about 10 mol. % POPC, about 30 mol. % DOPE, about 30 mol. %        MoChol and about 30 mol. % CHEMS;    -   (c) about 10 mol. % POPC, about 30 mol. % DOPE, about 20 mol. %        MoChol and about 40 mol. % CHEMS;    -   (d) about 6 mol. % POPC, about 24 mol. % DOPE, about 47 mol. %        MoChol and about 23 mol. % CHEMS.

Alternatively, said lipid phase may comprise a mixture of anionic andcationic lipids with amphoteric properties a neutral phosphatidylcholineand cholesterol. Such liposomes may also be serum-stable. In someembodiments, said lipid phase may comprise from 30 mol. % to 50 mol. %cholesterol, preferably from about 35 mol. % to about 45 mol. %.Alternatively, said lipid phase may comprise phosphatidylcholine andfrom 10 mol. % to 25 mol. % cholesterol, preferably from about 15 mol. %to about 25 mol. %.

A presently preferred formulation comprises 10 to 25 mol. % amphotericlipid, e.g. HistChol, HistDG or Acylcarnosin, 15 to 25 mol. %cholesterol and the remainder being POPC, soy bean PC, egg PC, DMPC,DPPC or DOPC, preferably POPC; for example about 60 mol. % POPC, about20 mol. % HistChol and about 20 mol. % Chol.

Another presently preferred formulation in accordance with the presentinvention comprises a liposome including a mix of lipid components withamphoteric properties and having about 30 mol. % POPC, about 10 mol. %DOTAP, about 20 mol. % CHEMS and about 40 mol. % Chol.

The amphoteric liposomes may have a size in the range 50 to 500 nm,preferably 100 to 500 nm, more preferably 150 and 300 nm.

The amphoteric liposome formulations of the present invention may beformulated for use as a colloid in a suitable pharmacologicallyacceptable vehicle. Vehicles such as water, saline, phosphate bufferedsaline and the like are well known to those skilled in the art for thispurpose.

In some embodiments, the amphoteric liposome formulations of the presentinvention may be administered at a physiological pH of between about 7and about 8. To this end, the formulation comprising the antisensecompound, excipient and vehicle may be formulated to have a pH in thisrange.

The amphoteric liposome formulations of the invention may bemanufactured using suitable methods that are known to those skilled inthe art. Such methods include, but are not limited to, extrusion throughmembranes of defined pore size, injection of lipid solutions in ethanolinto a water phase containing the cargo to be encapsulated, or highpressure homogenisation.

A solution of the oligonucleotide may be contacted with said excipientat a neutral pH, thereby resulting in volume inclusion of a certainpercentage of the solution. An high concentrations of the excipient,ranging from about 50 mM to about 150 mM, is preferred to achievesubstantial encapsulation of the active agent.

Amphoteric liposomes used as formulations in accordance with the presentinvention offer the distinct advantage of binding oligonucleotides at orbelow their isoelectric point, thereby concentrating said active agentat the liposome surface. This process is described in more detail in WO02/066012.

Irrespective of the actual production process used to make theamphoteric liposome formulations, in some embodiments, non-encapsulatedoligonucleotide may be removed from the liposomes after the initialproduction step in which the liposomes are formed as tight containers.Again, the technical literature and the references included hereindescribe such methodology in detail and suitable process steps mayinclude, but are not limited to, size exclusion chromatography,sedimentation, dialysis, ultrafiltration and diafiltration.

However, the removal of any non-encapsulated oligonucleotide is notrequired for performance of the invention, and in some embodiments thecomposition may comprise free as well as entrapped drug.

In some aspects of the invention the amphoteric liposome formulationswhich include the inventive antisense compounds may be used aspharmaceutical compositions for the prevention or treatment of aninflammatory, immune or autoimmune disorder of a human or non-humananimal such as graft rejection, graft-versus-host disease, multiplesclerosis, systemic lupus erythematosous, rheumatoid arthritis, asthma,inflammatory bowel disease, psoriasis or thyroiditis, Morbus Crohn andColitis ulcerosa.

Glossary Of Common Abbreviated Lipid Names

-   DMPC Dimyristoylphosphatidylcholine-   DPPC Dipalmitoylphosphatidylcholine-   DSPC Distearoylphosphatidylcholine-   POPC Palmitoyl-oleoylphosphatidylcholine-   DOPC Dioleoylphosphatidylcholine-   DOPE Dioleoylphosphatidylethanolamine-   DMPE Dimyristoylphosphatidylethanolamine-   DPPE Dipalmitoylphosphatidylethanolamine-   DOPG Dioleoylphosphatidylglycerol-   POPG Palmitoyl-oleoylphosphatidylglycerol-   DMPG Dimyristoylphosphatidylglycerol-   DPPG Dipalmitoylphosphatidylglycerol-   DMPS Dimyristoylphosphatidylserine-   DPPS Dipalmitoylphosphatidylserine-   DOPS Dioleoylphosphatidylserine-   POPS Palmitoyl-oleoylphosphatidylserine-   DMPA Dimyristoylphosphatidic acid-   DPPA Dipalmitoylphosphatidic acid-   DOPA Dioleoylphosphatidic acid-   POPA Palmitoyl-oleoylphosphatidic acid-   CHEMS Cholesterolhemisuccinate-   DC-Chol 3-β-[N—(N′,N′-dimethylethane) carbamoyl]cholesterol-   CetylP Cetylphosphate-   DODAP (1,2)-dioleoyloxypropyl)-N,N-dimethylammonium chloride-   DOEPC 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine-   DAC-Chol 3-β-[N—(N,N′-dimethylethane) carbamoyl]cholesterol-   TC-Chol 3-β-[N—(N′,N′,N′-trimethylaminoethane)carbamoyl]cholesterol-   DOTMA (1,2-dioleyloxypropyl)-N,N,N-trimethylammoniumchlorid)    (Lipofectin®)-   DOGS ((C18)₂GlySper3⁺) N,N-dioctadecylamido-glycylspermine    (Transfectam®)-   CTAB Cetyl-trimethylammoniumbromide,-   CPyC Cetyl-pyridiniumchloride-   DOTAP (1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium salt-   DMTAP (1,2-dimyristoyloxypropyl)-N,N,N-trimethylammonium salt-   DPTAP (1,2-dipalmitoyloxypropyl)-N,N,N-trimethylammonium salt-   DOTMA (1,2-dioleyloxypropyl)-N,N,N-trimethylammonium chloride)-   DORIE (1,2-dioleyloxypropyl)-3 dimethylhydroxyethyl ammoniumbromide)-   DDAB Dimethyldioctadecylammonium bromide-   DPIM 4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole-   CHIM Histaminyl-Cholesterolcarbamate-   MoChol 4-(2-Aminoethyl)-Morpholino-Cholesterolhemisuccinate-   His Chol Histaminyl-Cholesterolhemisuccinate.-   HCChol Nα-Histidinyl-Cholesterolcarbamate-   HistChol Nα-Histidinyl-Cholesterol-hemisuccinate.-   AC Acylcarnosine, Stearyl- & Palmitoylcarnosine-   HistDG    1,2-Dipalmitoylglycerol-hemisuccinate-Nα-Histidinyl-hemisuccinate, &    Distearoyl-, Dimyristoyl-, Dioleoyl- or palmitoyl-oleoylderivatives-   IsoHistSuccDG 1,2-Dipalmitoylglycerol-Oα-Histidinyl-Nα-hemisuccinat,    & Distearoyl-, Dimyristoyl-, Dioleoyl- or    palmitoyl-oleoylderivatives-   DGSucc 1,2-Dipalmitoyglycerol-3-hemisuccinate & Distearoyl-,    Dimyristoyl-Dioleoyl- or palmitoyl-oleoyl-derivatives

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. In oneembodiment, the present invention employs various penetration enhancersto effect the efficient delivery of nucleic acids, particularlyoligonucleotides. In addition to aiding the diffusion of non-lipophilicdrugs across cell membranes, penetration enhancers also enhance thepermeability of lipophilic drugs. Penetration enhancers may beclassified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG); cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA) or amphoteric lipids or lipid mixtures wherein amixture of cationic and anionic lipids displays amphoteric properties.For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Topical formulations are describedin detail in U.S. patent application Ser. No. 09/315,298 filed on May20, 1999, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein in its entirety. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Oralformulations for oligonucleotides and their preparation are described indetail in U.S. application Ser. Nos. 09/108,673 (filed Jul. 1, 1998),09/315,298 (filed May 20, 1999) and 10/071,822, filed Feb. 8, 2002, eachof which is incorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more other activeagents which function by a non-antisense mechanism, such as for examplechemotherapeutic agents or antiinflammatory drugs. Examples of suchchemotherapeutic agents include but are not limited to cancerchemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin,doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide,ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,mitomycin C, actinomycin D, mithramycin, prednisone,hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine,hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine,chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).

Anti-inflammatory drugs, including but not limited to non-steroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Alternatively, compositions ofthe invention may contain two or more antisense compounds targeted todifferent regions of the same nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity or expressionof CD40 nucleic acids can be tested in vitro in a variety of cell types.Cell types used for such analyses are available from commercial vendors(e.g. American Type Culture Collection, Manassas, Va.; Zen-Bio, Inc.,Research Triangle Park, N.C.; Clonetics Corporation, Walkersville, Md.)and cells are cultured according to the vendor's instructions usingcommercially available reagents (e.g. Invitrogen Life Technologies,Carlsbad, Calif.). Illustrative cell types include, but are not limitedto, HepG2 cells, Hep3B cells, HuVEC cells, T24, A549, and primaryhepatocytes.

In vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisenseoligonucleotides, which can be modified appropriately for treatment withother antisense compounds.

In general, cells are treated with antisense oligonucleotides when thecells reach approximately 60-80% confluency in culture.

One reagent commonly used to introduce antisense oligonucleotides intocultured cells includes the cationic lipid transfection reagentLIPOFECTIN® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotidesare mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, Calif.)to achieve the desired final concentration of antisense oligonucleotideand a LIPOFECTIN® concentration that typically ranges 2 to 12 ug/mL per100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes LIPOFECTAMINE® (Invitrogen, Carlsbad, Calif.).Antisense oligonucleotide is mixed with LIPOFECTAMINE® in OPTI-MEM® 1reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve thedesired concentration of antisense oligonucleotide and a LIPOFECTAMINE®concentration that typically ranges 2 to 12 ug/mL per 100 nM antisenseoligonucleotide.

Cells are treated with antisense oligonucleotides by routine methods.Cells are typically harvested 16-24 hours after antisenseoligonucleotide treatment, at which time RNA or protein levels of targetnucleic acids are measured by methods known in the art and describedherein. In general, when treatments are performed in multiplereplicates, the data are presented as the average of the replicatetreatments.

The concentration of antisense oligonucleotide used varies from cellline to cell line. Methods to determine the optimal antisenseoligonucleotide concentration for a particular cell line are well knownin the art. Antisense oligonucleotides are typically used atconcentrations ranging from 1 nM to 300 nM.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.Methods of RNA isolation are well known in the art. RNA is preparedusing methods well known in the art, for example, using the TRIZOL®Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer'srecommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of a CD40 nucleic acid can be assayedin a variety of ways known in the art. For example, target nucleic acidlevels can be quantitated by, e.g., Northern blot analysis, competitivepolymerase chain reaction (PCR), or quantitative real-time PCR. RNAanalysis can be performed on total cellular RNA or poly(A)+ mRNA.Methods of RNA isolation are well known in the art. Northern blotanalysis is also routine in the art. Quantitative real-time PCR can beconveniently accomplished using the commercially available ABI PRISM®7600, 7700, or 7900 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels may be accomplished by quantitativereal-time PCR using the ABI PRISM® 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. Methods of quantitative real-time PCRare well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reversetranscriptase (RT) reaction, which produces complementary DNA (cDNA)that is then used as the substrate for the real-time PCR amplification.The RT and real-time PCR reactions are performed sequentially in thesame sample well. RT and real-time PCR reagents are obtained fromInvitrogen (Carlsbad, Calif.). RT, real-time-PCR reactions are carriedout by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR are normalizedusing either the expression level of a gene whose expression isconstant, such as GAPDH or by quantifying total RNA using RIBOGREEN®(Invitrogen, Inc. Carlsbad, Calif.). GAPDH expression is quantified byreal time PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RIBOGREEN®RNA quantification reagent (Invetrogen, Inc. Eugene, Oreg.). Methods ofRNA quantification by RIBOGREEN® are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR® 4000instrument (PE Applied Biosystems) is used to measure RIBOGREEN®fluorescence.

Probes and primers are designed to hybridize to a CD40 nucleic acid.Methods for designing real-time PCR probes and primers are well known inthe art, and may include the use of software such as PRIMER EXPRESS®Software (Applied Biosystems, Foster City, Calif.).

Analysis of Protein Levels

Antisense inhibition of CD40 nucleic acids can be assessed by measuringCD40 protein levels. Protein levels of CD40 can be evaluated orquantitated in a variety of ways well known in the art, such asimmunoprecipitation, Western blot analysis (immunoblotting),enzyme-linked immunosorbent assay (ELISA), quantitative protein assays,protein activity assays (for example, caspase activity assays),immunohistochemistry, immunocytochemistry or fluorescence-activated cellsorting (FACS). Antibodies directed to a target can be identified andobtained from a variety of sources, such as the MSRS catalog ofantibodies (Aerie Corporation, Birmingham, Mich.), or can be preparedvia conventional monoclonal or polyclonal antibody generation methodswell known in the art. Antibodies useful for the detection of human andrat CD40 are commercially available.

In vivo Testing of Antisense Compounds

Antisense compounds, for example, antisense oligonucleotides, are testedin animals to assess their ability to inhibit expression of CD40 andproduce phenotypic changes, such as changes in cell morphology over timeor treatment dose as well as changes in levels of cellular componentssuch as proteins, nucleic acids, hormones, cytokines, and eosinophils.Testing may be performed in normal animals, or in experimental diseasemodels. For administration to animals, antisense oligonucleotides areformulated in a pharmaceutically acceptable diluent, such asphosphate-buffered saline. Administration includes pulmonaryadministration, aerosol administration, topical administration, andparenteral routes of administration, such as intraperitoneal,intravenous, and subcutaneous. Calculation of antisense oligonucleotidedosage and dosing frequency is within the abilities of those skilled inthe art, and depends upon factors such as route of administration andanimal body weight. Following a period of treatment with antisenseoligonucleotides, RNA is isolated from liver tissue and changes in CD40nucleic acid expression are measured.

Certain Indications

In certain embodiments, the invention provides methods of treating anindividual comprising administering one or more pharmaceuticalcompositions of the present invention. In certain embodiments, theindividual has an inflammatory or hyperproliferative disorder. Incertain embodiments the invention provides methods for prophylacticallyreducing CD40 expression in an individual. Certain embodiments includetreating an individual in need thereof by administering to an individuala therapeutically effective amount of an antisense compound targeted toa CD40 nucleic acid.

In one embodiment, administration of a therapeutically effective amountof an antisense compound targeted to a CD40 nucleic acid is accompaniedby monitoring of eosinophils in an individual, to determine anindividual's response to administration of the antisense compound. Anindividual's response to administration of the antisense compound isused by a physician to determine the amount and duration of therapeuticintervention.

In one embodiment, administration of an antisense compound targeted to aCD40 nucleic acid results in reduction of CD40 expression by at least15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or99%, or a range defined by any two of these values. In some embodiments,administration of a CD40 antisense compound increases the measure by atleast 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95or 99%, or a range defined by any two of these values. In someembodiments, administration of a CD40 antisense compound decreases themeasure by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95 or 99%, or a range defined by any two of these values.

In certain embodiments pharmaceutical composition comprising anantisense compound targeted to CD40 is used for the preparation of amedicament for treating a patient suffering or susceptible to aninflammatory condition or a hyperproliferative disorder.

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC50s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 ugto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

Certain Combination Therapies

In certain embodiments, one or more pharmaceutical compositions of thepresent invention are co-administered with one or more otherpharmaceutical agents. In certain embodiments, such one or more otherpharmaceutical agents are designed to treat the same disease orcondition as the one or more pharmaceutical compositions of the presentinvention. In certain embodiments, such one or more other pharmaceuticalagents are designed to treat a different disease or condition as the oneor more pharmaceutical compositions of the present invention. In certainembodiments, such one or more other pharmaceutical agents are designedto treat an undesired effect of one or more pharmaceutical compositionsof the present invention. In certain embodiments, one or morepharmaceutical compositions of the present invention are co-administeredwith another pharmaceutical agent to treat an undesired effect of thatother pharmaceutical agent. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are administered at the same time. Incertain embodiments, one or more pharmaceutical compositions of thepresent invention and one or more other pharmaceutical agents areadministered at different times. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are prepared together in a singleformulation. In certain embodiments, one or more pharmaceuticalcompositions of the present invention and one or more otherpharmaceutical agents are prepared separately.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition of the presentinvention include steroids and/or chemotherapeutic agents. In certainsuch embodiments, pharmaceutical agents that may be co-administered witha pharmaceutical composition of the present invention include, but arenot limited to prednisone, corticosteroids, and paclitaxel. In certainsuch embodiments, the agent is administered prior to administration of apharmaceutical composition of the present invention. In certain suchembodiments, the agent is administered following administration of apharmaceutical composition of the present invention. In certain suchembodiments the agent is administered at the same time as apharmaceutical composition of the present invention. In certain suchembodiments the dose of a co-administered agent is the same as the dosethat would be administered if the agent was administered alone. Incertain such embodiments the dose of a co-administered agent is lowerthan the dose that would be administered if the agent was administeredalone. In certain such embodiments the dose of a co-administered agentis greater than the dose that would be administered if the agent wasadministered alone.

In certain embodiments, the co-administration of a second compoundenhances the effect of a first compound, such that co-administration ofthe compounds results in an effect that is greater than the effect ofadministering the first compound alone. In other embodiments, theco-administration results in effects that are additive of the effects ofthe compounds when administered alone. In other embodiments, theco-administration results in effects that are supra-additive of theeffects of the compounds when administered alone. In some embodiments,the first compound is an antisense compound. In some embodiments, thesecond compound is an antisense compound.

EXAMPLES

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the referencesrecited in the present application is incorporated herein by referencein its entirety.

Example 1 Antisense Inhibition of Human CD40 In Vitro

Antisense oligonucleotides targeted to a CD40 nucleic acid were testedfor their effects on CD40 mRNA in vitro. When cultured cells, grown in a96-well plate, reached 80% confluency, they were treated with 150 nMantisense oligonucleotide. After a treatment period of approximately 24hours, RNA was isolated from the cells and CD40 mRNA levels weremeasured by quantitative real-time PCR, as described herein. CD40 mRNAlevels were adjusted according to total RNA content as measured bynormalization to RIBOGREEN®. Results are presented as percent inhibitionof CD40, relative to untreated control cells in Table 1.

The antisense oligonucleotides were designed as 18-mers withphosphorothioate backbones (internucleoside linkages) throughout. “5′target site” indicates the 5′-most nucleotide which the antisenseoligonucleotide is targeted to SEQ ID NO: 1 (GENBANK® Accession NoX60592.1). Data are averages from three experiments.

TABLE 1Inhibition of human CD40 mRNA levels by fully phosphorothioate oligodeoxynucleotidesTarget SEQ Target Target SEQ Oligo ID Start Stop Target % ID ID NO SiteSite Region Sequence (5′ to 3′) Inhibition NO 18623 1 18 35 5′ UTRCCAGGCGGCAGGACCACT 31 5 18624 1 20 37 5′ UTR GACCAGGCGGCAGGACCA 28 618625 1 26 43 5′ UTR AGGTGAGACCAGGCGGCA 22 7 18626 1 48 65 AUGCAGAGGCAGACGAACCAT 0 8 18627 1 49 66 Coding GCAGAGGCAGACGAACCA 0 9 186281 73 90 Coding GCAAGCAGCCCCAGAGGA 0 10 18629 1 78 95 CodingGGTCAGCAAGCAGCCCCA 30 11 18630 1 84 101 Coding GACAGCGGTCAGCAAGCA 0 1218631 1 88 105 Coding GATGGACAGCGGTCAGCA 0 13 18632 1 92 109 CodingTCTGGATGGACAGCGGTC 0 14 18633 1 98 115 Coding GGTGGTTCTGGATGGACA 0 1518634 1 101 118 Coding GTGGGTGGTTCTGGATGG 0 16 18635 1 104 121 CodingGCAGTGGGTGGTTCTGGA 0 17 18636 1 152 169 Coding CACAAAGAACAGCACTGA 0 1818637 1 156 173 Coding CTGGCACAAAGAACAGCA 0 19 18638 1 162 179 CodingTCCTGGCTGGCACAAAGA 0 20 18639 1 165 182 Coding CTGTCCTGGCTGGCACAA 5 2118640 1 176 193 Coding CTCACCAGTTTCTGTCCT 0 22 18641 1 179 196 CodingTCACTCACCAGTTTCTGT 0 23 18642 1 185 202 Coding GTGCAGTCACTCACCAGT 0 2418643 1 190 207 Coding ACTCTGTGCAGTCACTCA 0 25 18644 1 196 213 CodingCAGTGAACTCTGTGCAGT 5 26 18645 1 205 222 Coding ATTCCGTTTCAGTGAACT 0 2718646 1 211 228 Coding GAAGGCATTCCGTTTCAG 9 28 18647 1 222 239 CodingTTCACCGCAAGGAAGGCA 0 29 18648 1 250 267 Coding CTCTGTTCCAGGTGTCTA 0 3018649 1 267 284 Coding CTGGTGGCAGTGTGTCTC 0 31 18650 1 286 303 CodingTGGGGTCGCAGTATTTGT 0 32 18651 1 289 306 Coding GGTTGGGGTCGCAGTATT 0 3318652 1 292 309 Coding CTAGGTTGGGGTCGCAGT 0 34 18653 1 318 335 CodingGGTGCCCTTCTGCTGGAC 20 35 18654 1 322 339 Coding CTGAGGTGCCCTTCTGCT 16 3618655 1 332 349 Coding GTGTCTGTTTCTGAGGTG 0 37 18656 1 334 351 CodingTGGTGTCTGTTTCTGAGG 0 38 18657 1 345 362 Coding ACAGGTGCAGATGGTGTC 0 3918658 1 348 365 Coding TTCACAGGTGCAGATGGT 0 40 18659 1 360 377 CodingGTGCCAGCCTTCTTCACA 6 41 18660 1 364 381 Coding TACAGTGCCAGCCTTCTT 8 4218661 1 391 408 Coding GGACACAGCTCTCACAGG 0 43 18662 1 395 412 CodingTGCAGGACACAGCTCTCA 0 44 18663 1 401 418 Coding GAGCGGTGCAGGACACAG 0 4518664 1 416 433 Coding AAGCCGGGCGAGCATGAG 0 46 18665 1 432 449 CodingAATCTGCTTGACCCCAAA 6 47 18666 1 446 463 Coding GAAACCCCTGTAGCAATC 0 4818667 1 452 469 Coding GTATCAGAAACCCCTGTA 0 49 18668 1 463 480 CodingGCTCGCAGATGGTATCAG 0 50 18669 1 468 485 Coding GCAGGGCTCGCAGATGGT 34 5118670 1 471 488 Coding TGGGCAGGGCTCGCAGAT 0 52 18671 1 474 491 CodingGACTGGGCAGGGCTCGCA 3 53 18672 1 490 507 Coding CATTGGAGAAGAAGCCGA 0 5418673 1 497 514 Coding GATGACACATTGGAGAAG 0 55 18674 1 500 517 CodingGCAGATGACACATTGGAG 0 56 18675 1 506 523 Coding TCGAAAGCAGATGACACA 0 5718676 1 524 541 Coding GTCCAAGGGTGACATTTT 8 58 18677 1 532 549 CodingCACAGCTTGTCCAAGGGT 0 59 18678 1 539 556 Coding TTGGTCTCACAGCTTGTC 0 6018679 1 546 563 Coding CAGGTCTTTGGTCTCACA 7 61 18680 1 558 575 CodingCTGTTGCACAACCAGGTC 19 62 18681 1 570 587 Coding GTTTGTGCCTGCCTGTTG 2 6318682 1 575 592 Coding GTCTTGTTTGTGCCTGCC 0 64 18683 1 590 607 CodingCCACAGACAACATCAGTC 0 65 18684 1 597 614 Coding CTGGGGACCACAGACAAC 0 6618685 1 607 624 Coding TCAGCCGATCCTGGGGAC 0 67 18686 1 621 638 CodingCACCACCAGGGCTCTCAG 23 68 18687 1 626 643 Coding GGGATCACCACCAGGGCT 0 6918688 1 657 674 Coding GAGGATGGCAAACAGGAT 0 70 18689 1 668 685 CodingACCAGCACCAAGAGGATG 0 71 18690 1 679 696 Coding TTTTGATAAAGACCAGCA 0 7218691 1 703 720 Coding TATTGGTTGGCTTCTTGG 0 73 18692 1 729 746 CodingGGGTTCCTGCTTGGGGTG 0 74 18693 1 750 767 Coding GTCGGGAAAATTGATCTC 0 7518694 1 754 771 Coding GATCGTCGGGAAAATTGA 0 76 18695 1 765 782 CodingGGAGCCAGGAAGATCGTC 0 77 18696 1 766 783 Coding TGGAGCCAGGAAGATCGT 0 7818697 1 780 797 Coding TGGAGCAGCAGTGTTGGA 0 79 18698 1 796 813 CodingGTAAAGTCTCCTGCACTG 0 80 18699 1 806 823 Coding TGGCATCCATGTAAAGTC 0 8118700 1 810 827 Coding CGGTTGGCATCCATGTAA 0 82 18701 1 834 851 CodingCTCTTTGCCATCCTCCTG 4 83 18702 1 861 878 Coding CTGTCTCTCCTGCACTGA 0 8418703 1 873 890 Stop GGTGCAGCCTCACTGTCT 0 85 18704 1 910 927 3′ UTRAACTGCCTGTTTGCCCAC 34 86 18705 1 954 971 3′ UTR CTTCTGCCTGCACCCCTG 0 8718706 1 976 993 3′ UTR ACTGACTGGGCATAGCTC 0 88

Example 2 Antisense Inhibition of Human CD40 In Vitro

Antisense oligonucleotides targeted to a CD40 nucleic acid were testedfor their effects on CD40 mRNA in vitro. T24 cells at a density of 7000cells per well in a 96-well plate were treated with 150 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and CD40 mRNA levels were measured byquantitative real-time PCR, as described herein. CD40 mRNA levels wereadjusted according to GAPDH content, a housekeeping gene. Results arepresented as percent inhibition of CD40, relative to untreated controlcells in Table 2.

The antisense oligonucleotides were designed as 4-10-4 gapmers, wherethe gap segment comprises 2′-deoxynucleotides and each wing segmentcomprises 2′-MOE nucleotides and 5-methylcytosine substitutions. Theantisense oligonucleotides comprise phosphorothioate backbones(internucleoside linkages) throughout. “5′ target site” indicates the5′-most nucleotide which the antisense oligonucleotide is targeted toSEQ ID NO: 1 (GENBANK® Accession No X60592.1). Data are averages fromthree experiments. “ND” indicates a value was not determined.

TABLE 2Inhibition of human CD40 mRNA levels by chimeric oligonucleotides having4-10-4 MOE wings and deoxy gap Target SEQ Target Target SEQ ID StartStop Target % ID OligoID NO Site Site Region Sequence (5′ to 3′)Inhibition NO 19211 1 18 35 5′ UTR CCAGGCGGCAGGACCACT 76 5 19212 1 20 375′ UTR GACCAGGCGGCAGGACCA 77 6 19213 1 26 43 5′ UTR AGGTGAGACCAGGCGGCA81 7 19214 1 48 65 AUG CAGAGGCAGACGAACCAT 24 8 19215 1 49 66 CodingGCAGAGGCAGACGAACCA 46 9 19216 1 73 90 Coding GCAAGCAGCCCCAGAGGA 66 1019217 1 78 95 Coding GGTCAGCAAGCAGCCCCA 75 11 19218 1 84 101 CodingGACAGCGGTCAGCAAGCA 67 12 19219 1 88 105 Coding GATGGACAGCGGTCAGCA 65 1319220 1 92 109 Coding TCTGGATGGACAGCGGTC 79 14 19221 1 98 115 CodingGGTGGTTCTGGATGGACA 81 15 19222 1 101 118 Coding GTGGGTGGTTCTGGATGG 58 1619223 1 104 121 Coding GCAGTGGGTGGTTCTGGA 74 17 19224 1 152 169 CodingCACAAAGAACAGCACTGA 40 18 19225 1 156 173 Coding CTGGCACAAAGAACAGCA 60 1919226 1 162 179 Coding TCCTGGCTGGCACAAAGA 10 20 19227 1 165 182 CodingCTGTCCTGGCTGGCACAA 24 21 19228 1 176 193 Coding CTCACCAGTTTCTGTCCT 22 2219229 1 179 196 Coding TCACTCACCAGTTTCTGT 41 23 19230 1 185 202 CodingGTGCAGTCACTCACCAGT 82 24 19231 1 190 207 Coding ACTCTGTGCAGTCACTCA 38 2519232 1 196 213 Coding CAGTGAACTCTGTGCAGT 40 26 19233 1 205 222 CodingATTCCGTTTCAGTGAACT 56 27 19234 1 211 228 Coding GAAGGCATTCCGTTTCAG 32 2819235 1 222 239 Coding TTCACCGCAAGGAAGGCA 61 29 19236 1 250 267 CodingCTCTGTTCCAGGTGTCTA 62 30 19237 1 267 284 Coding CTGGTGGCAGTGTGTCTC 70 3119238 1 286 303 Coding TGGGGTCGCAGTATTTGT 0 32 19239 1 289 306 CodingGGTTGGGGTCGCAGTATT 19 33 19240 1 292 309 Coding CTAGGTTGGGGTCGCAGT 36 3419241 1 318 335 Coding GGTGCCCTTCTGCTGGAC 79 35 19242 1 322 339 CodingCTGAGGTGCCCTTCTGCT 70 36 19243 1 332 349 Coding GTGTCTGTTTCTGAGGTG 63 3719244 1 334 351 Coding TGGTGTCTGTTTCTGAGG 43 38 19245 1 345 362 CodingACAGGTGCAGATGGTGTC 73 39 19246 1 348 365 Coding TTCACAGGTGCAGATGGT 48 4019247 1 360 377 Coding GTGCCAGCCTTCTTCACA 61 41 19248 1 364 381 CodingTACAGTGCCAGCCTTCTT 47 42 19249 1 391 408 Coding GGACACAGCTCTCACAGG 0 4319250 1 395 412 Coding TGCAGGACACAGCTCTCA 52 44 19251 1 401 418 CodingGAGCGGTGCAGGACACAG 50 45 19252 1 416 433 Coding AAGCCGGGCGAGCATGAG 32 4619253 1 432 449 Coding AATCTGCTTGACCCCAAA 0 47 19254 1 446 463 CodingGAAACCCCTGTAGCAATC 0 48 19255 1 452 469 Coding GTATCAGAAACCCCTGTA 36 4919256 1 463 480 Coding GCTCGCAGATGGTATCAG 65 50 19257 1 468 485 CodingGCAGGGCTCGCAGATGGT 75 51 19258 1 471 488 Coding TGGGCAGGGCTCGCAGAT 0 5219259 1 474 491 Coding GACTGGGCAGGGCTCGCA 82 53 19260 1 490 507 CodingCATTGGAGAAGAAGCCGA 41 54 19261 1 497 514 Coding GATGACACATTGGAGAAG 14 5519262 1 500 517 Coding GCAGATGACACATTGGAG 78 56 19263 1 506 523 CodingTCGAAAGCAGATGACACA 59 57 19264 1 524 541 Coding GTCCAAGGGTGACATTTT 71 5819265 1 532 549 Coding CACAGCTTGTCCAAGGGT 0 59 19266 1 539 556 CodingTTGGTCTCACAGCTTGTC 46 60 19267 1 546 563 Coding CAGGTCTTTGGTCTCACA 64 6119268 1 558 575 Coding CTGTTGCACAACCAGGTC 82 62 19269 1 570 587 CodingGTTTGTGCCTGCCTGTTG 70 63 19270 1 575 592 Coding GTCTTGTTTGTGCCTGCC 69 6419271 1 590 607 Coding CCACAGACAACATCAGTC 11 65 19272 1 597 614 CodingCTGGGGACCACAGACAAC 9 66 19273 1 607 624 Coding TCAGCCGATCCTGGGGAC 0 6719274 1 621 638 Coding CACCACCAGGGCTCTCAG 23 68 19275 1 626 643 CodingGGGATCACCACCAGGGCT 58 69 19276 1 657 674 Coding GAGGATGGCAAACAGGAT 49 7019277 1 668 685 Coding ACCAGCACCAAGAGGATG ND 71 19278 1 679 696 CodingTTTTGATAAAGACCAGCA 31 72 19279 1 703 720 Coding TATTGGTTGGCTTCTTGG 49 7319280 1 729 746 Coding GGGTTCCTGCTTGGGGTG 14 74 19281 1 750 767 CodingGTCGGGAAAATTGATCTC 55 75 19282 1 754 771 Coding GATCGTCGGGAAAATTGA 0 7619283 1 765 782 Coding GGAGCCAGGAAGATCGTC 69 77 19284 1 766 783 CodingTGGAGCCAGGAAGATCGT 54 78 19285 1 780 797 Coding TGGAGCAGCAGTGTTGGA 15 7919286 1 796 813 Coding GTAAAGTCTCCTGCACTG 31 80 19287 1 806 823 CodingTGGCATCCATGTAAAGTC 65 81 19288 1 810 827 Coding CGGTTGGCATCCATGTAA 34 8219289 1 834 851 Coding CTCTTTGCCATCCTCCTG 42 83 19290 1 861 878 CodingCTGTCTCTCCTGCACTGA 26 84 19291 1 873 890 Stop GGTGCAGCCTCACTGTCT 76 8519292 1 910 927 3′ UTR AACTGCCTGTTTGCCCAC 63 86 19293 1 954 971 3′ UTRCTTCTGCCTGCACCCCTG 0 87 19294 1 976 993 3′ UTR ACTGACTGGGCATAGCTC 12 88

Example 3 Antisense Inhibition of Human CD40

Antisense oligonucleotides targeted to a CD40 nucleic acid were testedfor their effects on CD40 mRNA in vitro. T24 cells at a density of 7000cells per well in a 96-well plate were treated with 100 nM of antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and CD40 mRNA levels were measured byquantitative real-time PCR, as described herein. CD40 mRNA levels wereadjusted according to GAPDH content, a housekeeping gene. Results arepresented as percent inhibition of CD40, relative to untreated controlcells in Table 3.

The antisense oligonucleotides were designed as 4-10-4 gapmers, wherethe gap segment comprises 2′-deoxynucleotides and each wing segmentcomprises 2′-MOE nucleotides. The antisense oligonucleotides comprisephosphorothioate backbones (internucleoside linkages) and5-methylcytosine substitutions throughout. “5′ target site” indicatesthe 5′-most nucleotide which the antisense oligonucleotide is targetedto SEQ ID NO: 1 (GENBANK Accession No. X60592.1), SEQ ID NO: 2 (GENBANK®Accession No. H50598.1), and SEQ ID NO: 3 (GENBANK® Accession No.AA203290.1).

TABLE 3Inhibition of human CD40 mRNA levels by chimeric oligonucleotides having4-10-4 MOE wings and deoxy gap Target SEQ Target Target SEQ Oligo IDStart Stop % ID ID NO Site Site Sequence (5′ to 3′) Inhibition NO 261621 66 83 GCCCCAGAGGACGCACTG 0 89 26163 1 70 87 AGCAGCCCCAGAGGACGC 98 9026164 1 74 91 AGCAAGCAGCCCCAGAGG 47 91 26165 1 80 97 GCGGTCAGCAAGCAGCCC54 92 26167 1 95 112 GGTTCTGGATGGACAGCG 66 93 26168 1 102 119AGTGGGTGGTTCTGGATG 26 94 26169 1 141 158 GCACTGACTGTTTATTAG 43 95 261701 154 171 GGCACAAAGAACAGCACT 53 96 26171 1 164 181 TGTCCTGGCTGGCACAAA 2997 26172 1 171 188 CAGTTTCTGTCCTGGCTG 48 98 26173 1 180 197GTCACTCACCAGTTTCTG 47 99 26174 1 210 227 AAGGCATTCCGTTTCAGT 57 100 261751 224 241 CTTTCACCGCAAGGAAGG 34 101 26176 1 250 267 CTCTGTTCCAGGTGTCTA78 30 26177 1 257 274 TGTGTCTCTCTGTTCCAG 57 102 26178 1 264 281GTGGCAGTGTGTCTCTCT 0 103 26179 1 314 331 CCCTTCTGCTGGACCCGA 58 104 261801 321 338 TGAGGTGCCCTTCTGCTG 69 105 26181 1 329 346 TCTGTTTCTGAGGTGCCC44 106 26182 1 336 353 GATGGTGTCTGTTTCTGA 12 107 26183 1 364 381TACAGTGCCAGCCTTCTT 14 42 26184 1 445 462 AAACCCCTGTAGCAATCT 15 108 261851 460 477 CGCAGATGGTATCAGAAA 53 109 26186 1 469 486 GGCAGGGCTCGCAGATGG79 110 26202 1 485 502 GAGAAGAAGCCGACTGGG 0 111 26187 1 487 504TGGAGAAGAAGCCGACTG 23 112 26204 1 489 506 ATTGGAGAAGAAGCCGAC 0 113 262051 491 508 ACATTGGAGAAGAAGCCG 4 114 26206 1 493 510 ACACATTGGAGAAGAAGC 0115 26207 1 495 512 TGACACATTGGAGAAGAA 46 116 26188 1 496 513ATGACACATTGGAGAAGA 0 117 26208 1 497 514 GATGACACATTGGAGAAG 0 55 26189 1503 520 AAAGCAGATGACACATTG 6 118 26209 1 524 541 GTCCAAGGGTGACATTTT 5358 26210 1 545 562 AGGTCTTTGGTCTCACAG 81 119 26211 1 555 572TTGCACAACCAGGTCTTT 48 120 26212 1 570 587 GTTTGTGCCTGCCTGTTG 76 63 262131 572 589 TTGTTTGTGCCTGCCTGT 50 121 26214 1 574 591 TCTTGTTTGTGCCTGCCT87 122 26215 1 576 593 AGTCTTGTTTGTGCCTGC 83 123 26216 1 577 594CAGTCTTGTTTGTGCCTG 80 124 26217 1 578 595 TCAGTCTTGTTTGTGCCT 88 12526218 1 580 597 CATCAGTCTTGTTTGTGC 52 126 26219 1 590 607CCACAGACAACATCAGTC 16 65 26220 1 592 609 GACCACAGACAACATCAG 11 127 262211 594 611 GGGACCACAGACAACATC 40 128 26222 1 622 639 TCACCACCAGGGCTCTCA37 129 26223 1 624 641 GATCACCACCAGGGCTCT 82 130 26224 1 658 675AGAGGATGGCAAACAGGA 33 131 26225 1 659 676 AAGAGGATGGCAAACAGG 0 132 262261 660 677 CAAGAGGATGGCAAACAG 0 133 26227 1 669 686 GACCAGCACCAAGAGGAT 57134 26228 1 671 688 AAGACCAGCACCAAGAGG 35 135 26229 1 673 690TAAAGACCAGCACCAAGA 13 136 26230 1 676 693 TGATAAAGACCAGCACCA 0 137 262311 678 695 TTTGATAAAGACCAGCAC 26 138 26232 2 375 392 ACTCTCTTTGCCCATCCT 0139 26233 2 377 394 CGACTCTCTTTGCCCATC 31 140 26234 2 380 397ATGCGACTCTCTTTGCCC 12 141 26235 2 382 399 AAATGCGACTCTCTTTGC 36 14226236 2 385 402 CTGAAATGCGACTCTCTT 51 143 26237 2 387 404AACTGAAATGCGACTCTC 0 144 26238 2 406 423 CTTCACTGTCTCTCCCTG 0 145 262392 407 424 CCTTCACTGTCTCTCCCT 56 146 26240 2 409 426 AACCTTCACTGTCTCTCC 0147 26190 3 520 537 GATCACCACAGGCTCTCA 0 148 26191 3 565 582TGATAAGACAGCACCAAG 9 149 26192 3 584 601 GGTAGTTCTTGCCACTTT 0 150 261933 593 610 GGGCCTATGGGTAGTTCT 0 151 26194 3 617 634 ATTATCTCTGGGTCTGCT 9152 26195 3 646 663 ACTGACACATTTGAGCAG 0 153 26196 3 654 671GACTCCCTACTGACACAT 0 154 26197 3 689 706 CAAAGAGCGGTTCTCCAC 0 155 261983 696 713 AATTCTCCAAAGAGCGGT 0 156 26199 3 728 745 TCTTGACATCCTTTTCAT 0157 26200 3 736 753 CCCACCTATCTTGACATC 0 158 26201 3 791 808AGGCCGAGAGTTCAAAAT 0 159

Example 4 Antisense Inhibition of Human CD40

Antisense oligonucleotides targeted to a CD40 nucleic acid were testedfor their effects on CD40 mRNA in vitro. A549 cells at a density of 5000cells per well in a 96-well plate were treated with 120 nM of antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and CD40 mRNA levels were measured byquantitative real-time PCR, as described herein. CD40 primer probe setLTS37 was used to measure mRNA levels. CD40 mRNA levels were adjustedaccording to total RNA content as measured by RIBOGREEN®. Results arepresented as percent inhibition of CD40, relative to untreated controlcells in Table 4.

The antisense oligonucleotides were designed as 4-10-4 gapmers, 5-10-5gapmers, or 2-15-3 gapmers, where the gap segment comprises2′-deoxynucleotides and each wing segment comprises 2′-MOE nucleotides.The motif for each compound is indicated by the column labeled “motif.”The antisense oligonucleotides comprise phosphorothioate backbones(internucleoside linkages) and 5-methylcytosine substitutionsthroughout. “5′ target site” indicates the 5′-most nucleotide which theantisense oligonucleotide is targeted to SEQ ID NO: 1 (GENBANK AccessionNo. X60592.1) or SEQ ID NO: 4 (nucleotides 9797000 to 9813000 of GENBANKAccession No. NT_011362.9).

TABLE 4Inhibition of human CD40 mRNA levels by chimeric oligonucleotides having4-10-4 MOE wings and deoxy gap, 5-10-5 MOE wings and deoxy gap, and2-15-3 MOE wings and deoxy gap Target Target Target Oligo SEQ ID StartStop % SEQ ID ID NO Motif Site Site Sequence (5′ to 3′) Inhibition NO26163 4 4-10-4 2914 2931 AGCAGCCCCAGAGGACGC 74 90 396243 4 5-10-5 27282747 CCAGCAATTCACCGCGCAGG 0 160 396320 4 2-15-3 2728 2747CCAGCAATTCACCGCGCAGG 0 160 396199 4 5-10-5 2892 2911TGCAGAGGCAGACGAACCAT 75 161 396276 4 2-15-3 2892 2911TGCAGAGGCAGACGAACCAT 55 161 396200 4 5-10-5 2904 2923CAGAGGACGCACTGCAGAGG 79 162 396277 4 2-15-3 2904 2923CAGAGGACGCACTGCAGAGG 69 162 396201 4 5-10-5 2913 2932AAGCAGCCCCAGAGGACGCA 76 163 396278 4 2-15-3 2913 2932AAGCAGCCCCAGAGGACGCA 78 163 396202 4 5-10-5 2924 2943CAGCGGTCAGCAAGCAGCCC 68 164 396279 4 2-15-3 2924 2943CAGCGGTCAGCAAGCAGCCC 88 164 396244 4 5-10-5 2928 2947CTCACAGCGGTCAGCAAGCA 86 165 396321 4 2-15-3 2928 2947CTCACAGCGGTCAGCAAGCA 75 165 396245 4 5-10-5 3349 3368GCTGGCAAGGAGATGATAAC 51 166 396322 4 2-15-3 3349 3368GCTGGCAAGGAGATGATAAC 54 166 396246 4 5-10-5 3480 3499AGGTTGGAACACCCAAGATA 69 167 396323 4 2-15-3 3480 3499AGGTTGGAACACCCAAGATA 78 167 396247 4 5-10-5 3649 3668GGAGAAACCCCTGGTTTCTC 45 168 396324 4 2-15-3 3649 3668GGAGAAACCCCTGGTTTCTC 26 168 396248 4 5-10-5 3860 3879TCATTCCTGCCCAGGCTTCA 43 169 396325 4 2-15-3 3860 3879TCATTCCTGCCCAGGCTTCA 39 169 396249 4 5-10-5 3950 3969TCAGGTGAAAGTGAAAGCTG 68 170 396326 4 2-15-3 3950 3969TCAGGTGAAAGTGAAAGCTG 69 170 396250 4 5-10-5 4490 4509TACCATCTTCAAACACATGA 79 171 396327 4 2-15-3 4490 4509TACCATCTTCAAACACATGA 71 171 396251 4 5-10-5 4604 4623TTACCCAAAATGGGAAAGGA 86 172 396328 4 2-15-3 4604 4623TTACCCAAAATGGGAAAGGA 48 172 396252 4 5-10-5 4810 4829GAAAGAATACATGTATATGG 72 173 396329 4 2-15-3 4810 4829GAAAGAATACATGTATATGG 10 173 396253 4 5-10-5 4944 4963AGAGTCAGACAGCTTTAGAC 78 174 396330 4 2-15-3 4944 4963AGAGTCAGACAGCTTTAGAC 79 174 396254 4 5-10-5 5651 5670GTACCACCCATGCTATTAAT 79 175 396331 4 2-15-3 5651 5670GTACCACCCATGCTATTAAT 84 175 396255 4 5-10-5 5740 5759ACAGTGACAGAGTCCAAATG 85 176 396332 4 2-15-3 5740 5759ACAGTGACAGAGTCCAAATG 75 176 396256 4 5-10-5 5830 5849AATGTAAAGCTGGAAGGGTA 52 177 396333 4 2-15-3 5830 5849AATGTAAAGCTGGAAGGGTA 37 177 396257 4 5-10-5 5964 5983GGGCTATGTTTAGCACTTGG 79 178 396334 4 2-15-3 5964 5983GGGCTATGTTTAGCACTTGG 73 178 396258 4 5-10-5 6078 6097GGGCTTGATGCCTGAGTCAT 73 179 396335 4 2-15-3 6078 6097GGGCTTGATGCCTGAGTCAT 40 179 396259 4 5-10-5 6251 6270TGAAGTGCAAGTCAAAACAG 52 180 396336 4 2-15-3 6251 6270TGAAGTGCAAGTCAAAACAG 44 180 396260 4 5-10-5 6332 6351GCAATTTGAAGGGATCTTGA 68 181 396337 4 2-15-3 6332 6351GCAATTTGAAGGGATCTTGA 42 181 396203 4 5-10-5 6374 6393CATGCAGTGGGTGGTTCTGG 77 182 396280 4 2-15-3 6374 6393CATGCAGTGGGTGGTTCTGG 83 182 396204 4 5-10-5 6385 6404GTTTTTCTCTGCATGCAGTG 78 183 396281 4 2-15-3 6385 6404GTTTTTCTCTGCATGCAGTG 70 183 396205 4 5-10-5 6424 6443GCTGGCACAAAGAACAGCAC 61 184 396282 4 2-15-3 6424 6443GCTGGCACAAAGAACAGCAC 65 184 396261 4 5-10-5 6709 6728CACTAACCACACAATGATCA 85 185 396338 4 2-15-3 6709 6728CACTAACCACACAATGATCA 62 185 396206 4 5-10-5 6787 6806TGTGCAGTCACTCACCAGTT 83 186 396283 4 2-15-3 6787 6806TGTGCAGTCACTCACCAGTT 72 186 396207 4 5-10-5 6838 6857GTCTAGGAATTCGCTTTCAC 95 187 396284 4 2-15-3 6838 6857GTCTAGGAATTCGCTTTCAC 85 187 396208 4 5-10-5 6843 6862CAGGTGTCTAGGAATTCGCT 98 188 396285 4 2-15-3 6843 6862CAGGTGTCTAGGAATTCGCT 90 188 396209 4 5-10-5 6883 6902GTCGCAGTATTTGTGCTGGT 84 189 396286 4 2-15-3 6883 6902GTCGCAGTATTTGTGCTGGT 86 189 396262 4 5-10-5 7154 7173ACCCGAAGCCCTAGGTCTGA 92 190 396339 4 2-15-3 7154 7173ACCCGAAGCCCTAGGTCTGA 84 190 396210 4 5-10-5 7158 7177CTGGACCCGAAGCCCTAGGT 82 191 396287 4 2-15-3 7158 7177CTGGACCCGAAGCCCTAGGT 90 191 396211 4 5-10-5 7163 7182TTCTGCTGGACCCGAAGCCC 65 192 396288 4 2-15-3 7163 7182TTCTGCTGGACCCGAAGCCC 80 192 396212 4 5-10-5 7204 7223CTTCTTCACAGGTGCAGATG 79 193 396289 4 2-15-3 7204 7223CTTCTTCACAGGTGCAGATG 72 193 396263 4 5-10-5 7590 7609AGCCAGTGGCCAGGCAGGAC 70 194 396340 4 2-15-3 7590 7609AGCCAGTGGCCAGGCAGGAC 56 194 396214 4 5-10-5 7704 7723GAAGAAGCCGACTGGGCAGG 76 195 396291 4 2-15-3 7704 7723GAAGAAGCCGACTGGGCAGG 80 195 396215 4 5-10-5 7709 7728TTGGAGAAGAAGCCGACTGG 77 196 396292 4 2-15-3 7709 7728TTGGAGAAGAAGCCGACTGG 80 196 396216 4 5-10-5 7718 7737GATGACACATTGGAGAAGAA 76 197 396293 4 2-15-3 7718 7737GATGACACATTGGAGAAGAA 65 197 396264 4 5-10-5 7953 7972TGTCTATTACCTCAAAGAGA 89 198 396341 4 2-15-3 7953 7972TGTCTATTACCTCAAAGAGA 72 198 396265 4 5-10-5 8492 8511ACAGTGTGTTCAGAGGATTG 82 199 396342 4 2-15-3 8492 8511ACAGTGTGTTCAGAGGATTG 67 199 396266 4 5-10-5 9755 9774ACAATACACTTTACATGTTT 90 200 396343 4 2-15-3 9755 9774ACAATACACTTTACATGTTT 63 200 396267 4 5-10-5 10414 10433 ATTGTGTCTTTAGAACCAGA 84 201 396344 4 2-15-3 10414 10433 ATTGTGTCTTTAGAACCAGA 59 201 396268 4 5-10-5 10528 10547 GGGCCCTAAAGGATGTAAAA 34 202 396345 4 2-15-3 10528 10547 GGGCCCTAAAGGATGTAAAA 76 202 396217 4 5-10-5 11218 11237 CAGTCTTGTTTGTGCCTGCC 70 203 396294 4 2-15-3 11218 11237 CAGTCTTGTTTGTGCCTGCC 79 203 396269 4 5-10-5 11244 11263 TGTCCAGGACTCACCACAGA 77 204 396346 4 2-15-3 11244 11263 TGTCCAGGACTCACCACAGA 83 204 396270 4 5-10-5 11801 11820 TATGGCACCTTCTTAAATAT 85 205 396347 4 2-15-3 11801 11820 TATGGCACCTTCTTAAATAT 81 205 396271 4 5-10-5 12248 12267 TGCTTTTGGTATAGAAGAGT 86 206 396348 4 2-15-3 12248 12267 TGCTTTTGGTATAGAAGAGT 76 206 396235 4 5-10-5 12526 12545AAATGTGGCTGGCAGATGTC 79 207 396312 4 2-15-3 12526 12545AAATGTGGCTGGCAGATGTC 82 207 396236 4 5-10-5 12572 12591GTCAGAGCTCATCTACATCA 87 208 396313 4 2-15-3 12572 12591GTCAGAGCTCATCTACATCA 82 208 396237 4 5-10-5 12754 12773CTGATAAAGACCAGCACCAA 69 209 396314 4 2-15-3 12754 12773CTGATAAAGACCAGCACCAA 70 209 396238 4 5-10-5 12762 12781AGGACTCACTGATAAAGACC 69 210 396315 4 2-15-3 12762 12781AGGACTCACTGATAAAGACC 43 210 396239 4 5-10-5 12982 13001CAGACTCTGAATCAGTTTTA 78 211 396316 4 2-15-3 12982 13001CAGACTCTGAATCAGTTTTA 70 211 396240 4 5-10-5 13021 13040CAGTCCCCAATTCTGCTGCC 43 212 396317 4 2-15-3 13021 13040CAGTCCCCAATTCTGCTGCC 70 212 396241 4 5-10-5 13107 13126CCAGTGTTAGGCTCTGCCAG 76 213 396318 4 2-15-3 13107 13126CCAGTGTTAGGCTCTGCCAG 85 213 396242 4 5-10-5 13134 13153GAATGCCAGGAAAGGAGTGA 69 214 396319 4 2-15-3 13134 13153GAATGCCAGGAAAGGAGTGA 84 214 396272 4 5-10-5 13171 13190CAGCCCCAAGGCCCAAAGAT 48 215 396349 4 2-15-3 13171 13190CAGCCCCAAGGCCCAAAGAT 57 215 396220 4 5-10-5 13491 13510CTGCACTGGAGCAGCAGTGT 81 216 396297 4 2-15-3 13491 13510CTGCACTGGAGCAGCAGTGT 74 216 396221 4 5-10-5 13517 13536ACCGGTTGGCATCCATGTAA 61 217 396298 4 2-15-3 13517 13536ACCGGTTGGCATCCATGTAA 73 217 396222 4 5-10-5 13525 13544CCTGGGTGACCGGTTGGCAT 71 218 396299 4 2-15-3 13525 13544CCTGGGTGACCGGTTGGCAT 82 218 396223 4 5-10-5 13802 13821CAAGTTGGGAGACTGGATGG 65 219 396300 4 2-15-3 13802 13821CAAGTTGGGAGACTGGATGG 79 219 396224 4 5-10-5 13810 13829CTTTAATACAAGTTGGGAGA 68 220 396301 4 2-15-3 13810 13829CTTTAATACAAGTTGGGAGA 64 220 396225 4 5-10-5 13877 13896TCGGAAGGTCTGGTGGATAT 65 221 396302 4 2-15-3 13877 13896TCGGAAGGTCTGGTGGATAT 83 221 396226 4 5-10-5 13896 13915TGGGCACCAAACTGCTGGAT 70 222 396303 4 2-15-3 13896 13915TGGGCACCAAACTGCTGGAT 76 222 396227 4 5-10-5 13937 13956TATGGCTTCCTGGGCGCAGG 59 223 396304 4 2-15-3 13937 13956TATGGCTTCCTGGGCGCAGG 74 223 396228 4 5-10-5 13961 13980AATGCTGCAATGGGCATCTG 78 224 396305 4 2-15-3 13961 13980AATGCTGCAATGGGCATCTG 83 224 396229 4 5-10-5 13977 13996GTTCACTATCACAAACAATG 84 225 396306 4 2-15-3 13977 13996GTTCACTATCACAAACAATG 67 225 396230 4 5-10-5 13997 14016CAGTTAAGCAGCTTCCAGTT 84 226 396307 4 2-15-3 13997 14016CAGTTAAGCAGCTTCCAGTT 85 226 396231 4 5-10-5 14028 14047AATTTTATTTAGCCAGTCTC 80 227 396308 4 2-15-3 14028 14047AATTTTATTTAGCCAGTCTC 79 227 396232 4 5-10-5 14046 14065GTTGTATAAATATATTCTAA 44 228 396309 4 2-15-3 14046 14065GTTGTATAAATATATTCTAA 25 228 396233 4 5-10-5 14065 14084ACAGTGTTTTTGAGATTCTG 83 229 396310 4 2-15-3 14065 14084ACAGTGTTTTTGAGATTCTG 50 229 396273 4 5-10-5 14725 14744CTCAGGACCCAGAGTGAGGA 37 230 396350 4 2-15-3 14725 14744CTCAGGACCCAGAGTGAGGA 50 230 396274 4 5-10-5 15073 15092TGGGTTAAACCTCACCTCGA 59 231 396351 4 2-15-3 15073 15092TGGGTTAAACCTCACCTCGA 56 231 396275 4 5-10-5 15350 15369ATTAGGTCCCAAAGTTCCCC 23 232 396352 4 2-15-3 15350 15369ATTAGGTCCCAAAGTTCCCC 48 232 396234 1 5-10-5 42 61 GGCAGACGAACCATGGCGAG86 233 396311 1 2-15-3 42 61 GGCAGACGAACCATGGCGAG 82 233 396213 1 5-10-5435 454 GTAGCAATCTGCTTGACCCC 82 234 396290 1 2-15-3 435 454GTAGCAATCTGCTTGACCCC 79 234 396218 1 5-10-5 590 609 GACCACAGACAACATCAGTC89 235 396295 1 2-15-3 590 609 GACCACAGACAACATCAGTC 85 235 396219 15-10-5 683 702 CCACCTTTTTGATAAAGACC 65 236 396296 1 2-15-3 683 702CCACCTTTTTGATAAAGACC 41 236

Example 5 Antisense Inhibition of Human CD40 in HuVEC Cells, PrimerProbe Set LTS37

Several antisense oligonucleotides exhibiting in vitro inhibition ofCD40 (see Example 4) were tested at various doses in HuVEC cells. Cellswere plated at densities of 5000 cells per well and treated with nMconcentrations of antisense oligonucleotide as indicated in Table 5.After a treatment period of approximately 24 hours, RNA was isolatedfrom the cells and CD40 mRNA levels were measured by quantitativereal-time PCR, as described herein. Human CD40 primer probe set LTS37was used to measure mRNA levels. CD40 mRNA levels were adjustedaccording to total RNA content as measured by RIBOGREEN®. Results arepresented as percent inhibition of CD40, relative to untreated controlcells. As illustrated in Table 5, CD40 mRNA levels were reduced in adose-dependent manner.

TABLE 5 Antisense Inhibition of human CD40 in HuVEC cells, Primer ProbeSet LTS37 ISIS 0.2344 0.4688 0.9375 1.875 3.75 7.5 15.0 30.0 No nM nM nMnM nM nM nM nM 26163 17 35 38 51 62 67 82 89 396236 23 49 59 77 86 92 9189 396266 35 45 58 74 55 72 57 56 396307 21 45 43 56 80 79 82 82 39621834 47 52 57 78 82 86 86 396279 34 54 59 49 72 82 88 87 396287 31 48 5250 64 77 85 86 396264 39 34 49 56 71 84 88 86

Example 6 Antisense Inhibition of Human CD40 in AGS Cells

Antisense oligonucleotides exhibiting in vitro inhibition of CD40 (seeExample 4) were tested at various doses in AGS cells (humanadenocarcinoma cells). Antisense oligonucleotides of SEQ ID No. 90 andSEQ ID No. 208 were designed as 4-10-4 gapmers or 5-10-5 gapmers,respectively, where the gap segment comprises 2′-deoxynucleotides andeach wing segment comprises 2′-MOE or 2′OMe nucleotides. The antisenseoligonucleotides comprise phosphorothioate backbones (internucleosidelinkages) and 5-methylcytosine substitutions throughout.

Cells were plated at densities of 5000 cells per well and treated withnM concentrations of antisense oligonucleotide as indicated in Table 6.After a treatment period of approximately 24 hours, RNA was isolatedfrom the cells and relative CD40 mRNA expression levels were quantifiedby real time RT-PCR using the QuantiTect™ SYBR® Green RT-PCR kit(Qiagen). CD40 mRNA levels were adjusted according to GAPDH content, ahousekeeping gene. Results are presented as percent inhibition of CD40,relative to cells treated with a scrambled control oligonucleotide (TCCATTTATTAGTCTAGGAA (5-10-5 gapmer, where the gap segment comprises2′-deoxynucleotides and each wing segment comprises 2′-MOE nucleotides.The oligonucleotide comprises phosphorothioate backbones(internucleoside linkages) and 5-methylcytosine substitutionsthroughout.

TABLE 6 Antisense Inhibition of human CD40 in AGS cells ISIS Wing 12.525.0 50.0 No Motif segment nM nM nM Seq ID  26163 4-10-4 2′MOE 80 69 9090 396236 5-10-5 2′MOE 83 86 94 208 — 4-10-4 2′OMe 51 54 66 90 — 5-10-52′OMe 60 63 69 208As illustrated in Table 6, CD40 mRNA levels were reduced in adose-dependent manner. Antisense oligonucleotides comprising 2′MOE wingsegments are more active than those with 2′OMe wing segments.

Example 7 Antisense Inhibition of Murine CD40 In Vitro

Chimeric antisense oligonucleotides having 5-10-5 MOE wings and deoxygap and 4-10-4 MOE wings and deoxy gap may be designed to target murineCD40. These antisense oligonucleotides can be evaluated for theirability to reduce CD40 mRNA in primary mouse hepatocytes using similarmethods as described in the human in vitro study.

For example, primary mouse hepatocytes may be treated with 0.2344 nM,0.4688 nM, 0.9375 nM, 1.875 nM, 3.75 nM, 7.5 nM, 15.0 nM, and 30.0 nM ofantisense oligonucleotides for a period of approximately 24 hours. RNAcan be isolated from the cells and CD40 mRNA levels can be measured byquantitative real-time PCR, as described herein. Murine CD40 primerprobe sets can be used to measure mRNA levels. CD40 mRNA levels can thenbe adjusted according to total RNA content as measured by RIBOGREEN®.

Example 8 Antisense Inhibition of Murine CD40 In Vivo

Antisense oligonucleotides showing statistically significantdose-dependent inhibition from an in vitro study can be evaluated fortheir ability to reduce CD40 mRNA in vivo.

Treatment

Antisense oligonucleotide can be evaluated in Balb/c mice and comparedto a control group treated with saline. Oligonucleotide or saline wouldbe administered subcutaneously at a dose of 5 mg/kg, 10 mg/kg, 25 mg/kg,or 50 mg/kg twice a week for three weeks. After the treatment period,whole liver can be collected for RNA analysis and protein analysis.

RNA Analysis

Liver RNA can be isolated for real-time PCR analysis of CD40. It istheorized that an antisense oligonucleotide showing significantdose-dependent inhibition in vitro may show significant dose-dependentinhibition in vivo.

Protein Analysis

Liver CD40 protein may be measured by Western blot.

Example 9 Tolerability of Antisense Compounds in Rodents

Male 6 week old Balb/c mice were dosed subcutaneous 2× per week for 4weeks with 25 or 50 mg/kg of antisense oligonucleotides Isis 26163 orIsis 396236. Mice were sacrificed 2 days following last administration.Body weights of the animals were monitored throughout the study. Aftersacrification liver, spleen and kidney weights and liver enzymes ALT andAST from mouse plasma were determined.

Compared to a saline control treatment body weights of the mice are notaffected by antisense oligonucleotides Isis 26163 or Isis 396236. Liverweight and spleen weight displayed a slight increase for Isis 26163 butnot for Isis 396236. LFT (liver function test) elevations were small andwithin the normal range of high dose mouse studies.

Example 10 Antisense Inhibition of Human CD40 In Vitro on T24Cells—Comparative Data for ISIS 26163 and ISIS19216

Antisense oligonucleotides ISIS 26163 and ISIS19216 targeted to a CD40nucleic acid were tested for their effects on CD40 mRNA in vitro. Theantisense oligonucleotides were designed as 4-10-4 gapmers, where thegap segment comprises 2′-deoxynucleotides and each wing segmentcomprises 2′-MOE nucleotides. The antisense oligonucleotides comprisephosphorothioate backbones (internucleoside linkages) and5-methylcytosine substitutions throughout or in the wings, respectively.

T24 cells at a density of 7000 cells per well in a 96-well plate weretreated with 100 nM or 150 nM, respectively, of antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and CD40 mRNA levels were measured byquantitative real-time PCR, as described herein. CD40 mRNA levels wereadjusted according to GAPDH content, a housekeeping gene.

Results are presented in Table 7 as percent inhibition of CD40, relativeto untreated control cells.

TABLE 7 Oligo ID Target site nM on T24 cells % Inhibition SEQ ID No.26163 70-87 100 98 90 19216 73-90 150 66 10

Sequence ISIS 26163 shows a superior activity over ISIS19126, whichoverlaps the sequence 15 nucleobases.

The invention claimed is:
 1. A modified antisense compound 12 to 30nucleobases in length and having a nucleobase sequence that is at least90% complementary to an equal length portion of the human CD40 gene butnot to other sequences throughout the human genome, selected from thefollowing regions of SEQ ID NO: 4: (a) positions 11250-12685,corresponding to intron 6 11801-12591; (b) positions 2943-6367,corresponding to intron 1; (c) positions 6447-6780, corresponding tointron 2; (d) positions 6907-7157, corresponding to intron 3; (e)positions 7305-7673, corresponding to intron 4; (f) positions7768-11187, corresponding to intron 5; (g) positions 12773-12877,corresponding to intron 7; (h) positions 12907-13429, corresponding tointron 8; and (i) positions 13662-16001, which forms part of exon 9 or aregion 3′ to exon
 9. 2. The antisense compound of claim 1, wherein thenucleobase sequence is at least 90% complementary to an equal lengthportion of positions 12527-12685 of SEQ ID NO:
 4. 3. The antisensecompound of claim 2 1, having a nucleobase sequence comprising at least8 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 208,wherein the nucleobase sequence of the compound is at least 95%complementary to the sequence shown in SEQ ID NO:
 4. 4. A modifiedantisense compound 20 nucleobases in length and consisting of thenucleobase sequence of SEQ ID NO:
 208. 5. The antisense compound ofclaim 1, wherein said antisense compound is an antisenseoligonucleotide.
 6. The antisense compound of claim 5, wherein at leastone internucleoside linkage is a modified internucleoside linkage. 7.The antisense compound of claim 6, wherein each internucleoside linkageis a phosphorothioate internucleoside linkage.
 8. The antisense compoundof claim 5, wherein at least one nucleoside comprises a modified sugar.9. The antisense compound of claim 8, wherein at least one modifiedsugar is a bicyclic sugar.
 10. The antisense compound of claim 9,wherein the at least one bicyclic sugar comprises a 4′-CH(CH₃)—O-2′bridge.
 11. The antisense compound of claim 8, wherein at least onemodified sugar comprises a 2′-O-methoxyethyl.
 12. The antisense compoundof claim 1, wherein at least one said nucleobase is a modifiednucleobase.
 13. The antisense compound of claim 12, wherein the modifiednucleobase is a 5-methylcytosine.
 14. The antisense compound of claim 1,wherein the compound is an oligonucleotide comprising: a gap segmentconsisting of linked deoxynucleosides; a 5′ wing segment consisting oflinked nucleosides; a 3′ wing segment consisting of linked nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment and wherein each nucleoside of each wing segmentcomprises a modified sugar.
 15. The antisense compound of claim 14,wherein the oligonucleotide comprises: a gap segment consisting of tenlinked deoxynucleosides; a 5′ wing segment consisting of five linkednucleosides; a 3′ wing segment consisting of five linked nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar; and wherein each internucleosidelinkage of said antisense compound is a phosphorothioate linkage. 16.The antisense compound of claim 14, wherein the oligonucleotidecomprises: a gap segment consisting of fifteen linked deoxynucleosides;a 5′ wing segment consisting of two linked nucleosides; a 3′ wingsegment consisting of three linked nucleosides; wherein the gap segmentis positioned between the 5′ wing segment and the 3′ wing segment,wherein each nucleoside of each wing segment comprises a2′-O-methoxyethyl sugar; and wherein each internucleoside linkage ofsaid antisense compound is a phosphorothioate linkage.
 17. The antisensecompound of claim 15 or 16, wherein every cytosine is a5-methylcytosine.
 18. An antisense oligonucleotide 20 nucleobases inlength having the sequence of nucleobases as set forth in SEQ ID NO:208,wherein each cytosine is a 5-methylcytosine, each internucleosidelinkage is a phosphorothioate linkage, nucleotides 1-5 and 16-20 are2′-O-methoxyethyl nucleotides, and nucleotides 6-15 are2′-deoxynucleotides.
 19. A composition comprising an antisense compoundof claim 1 or 18 or a salt thereof and a pharmaceutically acceptablecarrier or diluent.
 20. A method comprising administering to an animalan antisense compound of claim 1 or an oligonucleotide of claim
 18. 21.The antisense compound of claim 1, wherein said antisense compound is15, 16, 17, 18, 19, 20 or 21 nucleobases in length.
 22. The antisensecompound of claim 21, wherein the nucleobase sequence of the compound isat least 95% complementary to the sequence shown in SEQ ID NO:
 4. 23.The antisense compound of claim 21, wherein the nucleobase sequence ofthe compound is 100% complementary to the sequence shown in SEQ ID NO:4.